Rna containing composition for treatment of tumor diseases

ABSTRACT

The present invention relates to RNA containing compositions for use in the treatment or prophylaxis of tumor and/or cancer diseases, to a pharmaceutical composition, to a kit and to uses of the RNA containing compositions for the treatment or prophylaxis of tumor and/or cancer diseases.

This application claims the priority of European Application No.15001191.4, filed Apr. 22, 2015, the entirety of which is incorporatedherein by reference.

The sequence listing that is contained in the file named“CRVCP0175US.txt”, which is 18,426 KB (as measured in Microsoft Windows)and was created on Apr. 22, 2016, is filed herewith by electronicsubmission and is incorporated herein by reference.

INTRODUCTION

The present invention relates to RNA containing compositions for use inthe treatment or prophylaxis of tumor and/or cancer diseases, to apharmaceutical composition, to a kit and to uses of the RNA containingcompositions for the treatment or prophylaxis of tumor and/or cancerdiseases.

Cancer, also known as malignant tumor, describes a group of diseasesinvolving abnormal cell growth with the potential to invade or spread toother parts of the body. In 2012, about 14.1 million new cases of canceroccurred globally (not including skin cancer other than melanoma).

The standard treatments of cancer include chemotherapy, radiation andsurgery, wherein these treatments are applied individually or incombination. Other treatments apply cancer immunotherapy which isfocused on stimulating the immune system through vaccination or adoptivecellular immunotherapy to elicit an anti-tumor response.

Some approaches use gene therapy and genetic vaccination for treatmentof cancer or other tumor diseases. Gene therapy and genetic vaccinationare molecular medicine methods which are based on the introduction ofnucleic acids into cells or into tissues of a patient. Subsequently theinformation coded by the nucleic acids introduced is processed in theorganism, i.e. resulting in expression of a therapeutic peptide orprotein or expression of an antigen which is coded by the nucleic acids.

Conventional gene therapeutic methods, including gene therapy andgenetic vaccination are based on the use of DNA molecules in order totransfer the desired genetic information into the cell. Various methodshave been developed for introducing DNA into cells, such as calciumphosphate transfection, polybrene transfection, protoplast fusion,electroporation, microinjection and lipofection. DNA viruses maylikewise be used as a DNA vehicle achieving a very high transfectionrate. The use of DNA entails the risk of the DNA being inserted into anintact gene of the host cell's genome by e.g. recombination. In thiscase the affected gene may be mutated and inactivated or may give riseto misinformation. Another risk of using DNA as a pharmaceutical agentis the risk of inducing pathogenic anti-drug antibodies (anti-DNAantibodies) in the patient, which may result in a (possibly fatal)immune response.

The use of RNA as a gene therapeutic agent or genetic vaccine issubstantially safer, because RNA does not involve the risk of beingintegrated into the genome inducing an undesired pathogenic induction ofanti-drug antibodies.

Thus RNA expression systems have considerable advantages over DNAexpression systems in gene therapy and in genetic vaccination althoughit is known in the prior art or rather assumed for a long time that theinstability of mRNA or of RNA in general may be problem in theapplication of medical methods based on RNA expression systems.

The instability of RNA is in particular due to RNA-degrading enzymes(ribonucleases-RNases). There are also many further processes whichdestabilize RNA, wherein interaction between the RNA and proteins oftenappears to play a crucial role. Some measures for increasing thestability of RNA have been proposed, so enabling the use thereof as agene therapy agent or RNA vaccine.

For solving the problem of ex vivo RNA stability the European patentapplication EP 1 083 232 A1 describes a method for introducing RNA, inparticular mRNA, into cells and organisms, in which the RNA forms acomplex with a cationic peptide or protein.

The application of mRNA is known for the treatment and/or prophylaxis ofcancer. For example the international patent application WO 03/051401 A2describes a pharmaceutical composition comprising at least one mRNA,which contains at least one region that codes for an antigen from atumor, combined with an aqueous solvent and preferably with a cytokinee.g. GM-CSF. The pharmaceutical composition is proposed to be used fortherapy and/or prophylaxis against cancer.

The international patent application WO 2006/008154 A1 discloses an mRNAmixture for vaccinating against tumor diseases, wherein at least onetype of mRNA contains at least one tumor antigen-coding region. At leastone other mRNA contains at least one type of an immunogenicprotein-coding region.

Nevertheless there is still a need for an effective treatment of tumordiseases and especially for the treatment of cancer. Therefore it is theobject of the underlying invention to provide an approach for effectivetreatment of tumor diseases wherein tumor tissue and cancer cells arespecifically destroyed.

This object is solved by the subject matter of the claims. Particularly,the object underlying the present invention is solved according to afirst aspect by an RNA containing composition for use in the treatmentor prophylaxis of tumor and/or cancer diseases. According to furtheraspects of the invention the object is solved by a pharmaceuticalcomposition, by a kit or kit of parts, and by a method of treatment oftumor or cancer diseases.

DEFINITIONS

For the sake of clarity and readability the following scientificbackground information and definitions are provided. Any technicalfeatures disclosed thereby can be part of each and every embodiment ofthe invention. Additional definitions and explanations can be providedin the context of this disclosure.

Immune system: The immune system may protect organisms from infection.If a pathogen breaks through a physical barrier of an organism andenters this organism, the innate immune system provides an immediate,but non-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained after the pathogen has been eliminated, in the form of animmunological memory, and allows the adaptive immune system to mountfaster and stronger attacks each time this pathogen is encountered.According to this, the immune system comprises the innate and theadaptive immune system. Each of these two parts contains so calledhumoral and cellular components.

Immune response: An immune response may typically either be a specificreaction of the adaptive immune system to a particular antigen (socalled specific or adaptive immune response) or an unspecific reactionof the innate immune system (so called unspecific or innate immuneresponse).

Adaptive immune system: The adaptive immune system is composed of highlyspecialized, systemic cells and processes that eliminate or preventpathogenic growth. The adaptive immune response provides the vertebrateimmune system with the ability to recognize and remember specificpathogens (to generate immunity), and to mount stronger attacks eachtime the pathogen is encountered. The system is highly adaptable becauseof somatic hypermutation (a process of increased frequency of somaticmutations), and V(D)J recombination (an irreversible geneticrecombination of antigen receptor gene segments). This mechanism allowsa small number of genes to generate a vast number of different antigenreceptors, which are then uniquely expressed on each individuallymphocyte. Because the gene rearrangement leads to an irreversiblechange in the DNA of each cell, all of the progeny (offspring) of thatcell will then inherit genes encoding the same receptor specificity,including the Memory B cells and Memory T cells that are the keys tolong-lived specific immunity. Immune network theory is a theory of howthe adaptive immune system works, that is based on interactions betweenthe variable regions of the receptors of T cells, B cells and ofmolecules made by T cells and B cells that have variable regions.

Adaptive immune response: The adaptive immune response is typicallyunderstood to be antigen-specific. Antigen specificity allows for thegeneration of responses that are tailored to specific antigens,pathogens or pathogen-infected cells. The ability to mount thesetailored responses is maintained in the body by “memory cells”. Should apathogen infect the body more than once, these specific memory cells areused to quickly eliminate it. In this context, the first step of anadaptive immune response is the activation of naïve antigen-specific Tcells or different immune cells able to induce an antigen-specificimmune response by antigen-presenting cells. This occurs in the lymphoidtissues and organs through which naïve T cells are constantly passing.Cell types that can serve as antigen-presenting cells are inter aliadendritic cells, macrophages, and B cells. Each of these cells has adistinct function in eliciting immune responses. Dendritic cells take upantigens by phagocytosis and macropinocytosis and are stimulated bycontact with e.g. a foreign antigen to migrate to the local lymphoidtissue, where they differentiate into mature dendritic cells.Macrophages ingest particulate antigens such as bacteria and are inducedby infectious agents or other appropriate stimuli to express MHCmolecules. The unique ability of B cells to bind and internalize solubleprotein antigens via their receptors may also be important to induce Tcells. Presenting the antigen on MHC molecules leads to activation of Tcells which induces their proliferation and differentiation into armedeffector T cells. The most important function of effector T cells is thekilling of infected cells by CD8+ cytotoxic T cells and the activationof macrophages by Th1 cells which together make up cell-mediatedimmunity, and the activation of B cells by both Th2 and Th1 cells toproduce different classes of antibody, thus driving the humoral immuneresponse. T cells recognize an antigen by their T cell receptors whichdo not recognize and bind antigen directly, but instead recognize shortpeptide fragments e.g. of pathogen-derived protein antigens, which arebound to MHC molecules on the surfaces of other cells.

Cellular immunity/cellular immune response: Cellular immunity relatestypically to the activation of macrophages, natural killer cells (NK),antigen-specific cytotoxic T-lymphocytes, and the release of variouscytokines in response to an antigen. In a more general way, cellularimmunity is not related to antibodies but to the activation of cells ofthe immune system. A cellular immune response is characterized e.g. byactivating antigen-specific cytotoxic T-lymphocytes that are able toinduce apoptosis in body cells displaying epitopes of an antigen ontheir surface, such as virus-infected cells, cells with intracellularbacteria, and cancer cells displaying tumor antigens; activatingmacrophages and natural killer cells, enabling them to destroypathogens; and stimulating cells to secrete a variety of cytokines thatinfluence the function of other cells involved in adaptive immuneresponses and innate immune responses.

Humoral immunity/humoral immune response: Humoral immunity referstypically to antibody production and the accessory processes that mayaccompany it. A humoral immune response may be typically characterized,e.g., by Th2 activation and cytokine production, germinal centerformation and isotype switching, affinity maturation and memory cellgeneration. Humoral immunity also typically may refer to the effectorfunctions of antibodies, which include pathogen and toxinneutralization, classical complement activation, and opsonin promotionof phagocytosis and pathogen elimination.

Innate immune system: The innate immune system, also known asnon-specific immune system, comprises the cells and mechanisms thatdefend the host from infection by other organisms in a non-specificmanner. This means that the cells of the innate system recognize andrespond to pathogens in a generic way, but unlike the adaptive immunesystem, it does not confer long-lasting or protective immunity to thehost. The innate immune system may be e.g. activated by ligands ofpathogen-associated molecular patterns (PAMP) receptors, e.g. Toll-likereceptors (TLRs) or other auxiliary substances such aslipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines,lymphokines, interleukins or chemokines, immunostimulatory nucleicacids, immunostimulatory RNA (isRNA), CpG-DNA, antibacterial agents, oranti-viral agents. Typically a response of the innate immune systemincludes recruiting immune cells to sites of infection, through theproduction of chemical factors, including specialized chemicalmediators, called cytokines; activation of the complement cascade;identification and removal of foreign substances present in organs,tissues, the blood and lymph, by specialized white blood cells;activation of the adaptive immune system through a process known asantigen presentation; and/or acting as a physical and chemical barrierto infectious agents.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in thebroadest sense is typically a (e.g. pharmacological or immunological)agent or composition that may modify, e.g. enhance, the efficacy ofother agents, such as a drug or vaccine. Conventionally the term refersin the context of the invention to a compound or composition that servesas a carrier or auxiliary substance for immunogens and/or otherpharmaceutically active compounds. It is to be interpreted in a broadsense and refers to a broad spectrum of substances that are able toincrease the immunogenicity of antigens incorporated into orco-administered with an adjuvant in question. In the context of thepresent invention an adjuvant will preferably enhance the specificimmunogenic effect of the active agents of the present invention.Typically, “adjuvant” or “adjuvant component” has the same meaning andcan be used mutually. Adjuvants may be divided, e.g., into immunopotentiators, antigenic delivery systems or even combinations thereof.

The term “adjuvant” is typically understood not to comprise agents whichconfer immunity by themselves. An adjuvant assists the immune systemunspecifically to enhance the antigen-specific immune response by e.g.promoting presentation of an antigen to the immune system or inductionof an unspecific innate immune response. Furthermore, an adjuvant maypreferably e.g. modulate the antigen-specific immune response by e.g.shifting the dominating Th2-based antigen specific response to a moreTh1-based antigen specific response or vice versa. Accordingly, anadjuvant may favourably modulate cytokine expression/secretion, antigenpresentation, type of immune response etc.

Immunostimulatory/immunostimulating RNA: Animmunostimulatory/immunostimulating RNA (isRNA) in the context of theinvention may typically be a RNA that is able to induce an innate immuneresponse itself. It usually does not have an open reading frame and thusdoes not provide a peptide-antigen or immunogen but elicits an innateimmune response e.g. by binding to a specific kind of Toll-like-receptor(TLR) or other suitable receptors. Thereforeimmunostimulatory/immunostimulating RNAs are preferably non-coding RNAs.However, of course also mRNAs having an open reading frame and codingfor a peptide/protein (e.g. an antigenic function) may induce an innateimmune response.

Antigen: The term “antigen” refers typically to a substance which may berecognized by the immune system and may be capable of triggering anantigen-specific immune response, e.g. by formation of antibodies orantigen-specific T-cells as part of an adaptive immune response. Anantigen may be a protein or peptide. In this context, the first step ofan adaptive immune response is the activation of naïve antigen-specificT cells by antigen-presenting cells. This occurs in the lymphoid tissuesand organs through which naïve T cells are constantly passing. The threecell types that can serve as antigen-presenting cells are dendriticcells, macrophages, and B cells. Each of these cells has a distinctfunction in eliciting immune responses. Tissue dendritic cells take upantigens by phagocytosis and macropinocytosis and are stimulated byinfection to migrate to the local lymphoid tissue, where theydifferentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents to express MHC class II molecules. The unique ability of B cellsto bind and internalize soluble protein antigens via their receptors maybe important to induce T cells. By presenting the antigen on MHCmolecules leads to activation of T cells which induces theirproliferation and differentiation into armed effector T cells. The mostimportant function of effector T cells is the killing of infected cellsby CD8+ cytotoxic T cells and the activation of macrophages by Th1 cellswhich together make up cell-mediated immunity, and the activation of Bcells by both Th2 and Th1 cells to produce different classes ofantibody, thus driving the humoral immune response. T cells recognize anantigen by their T cell receptors which does not recognize and bindantigen directly, but instead recognize short peptide fragments e.g. ofpathogens' protein antigens, which are bound to MHC molecules on thesurfaces of other cells.

T cells fall into two major classes that have different effectorfunctions. The two classes are distinguished by the expression of thecell-surface proteins CD4 and CD8. These two types of T cells differ inthe class of MHC molecule that they recognize. There are two classes ofMHC molecules—MHC class I and MHC class II molecules—which differ intheir structure and expression pattern on tissues of the body. CD4+ Tcells bind to a MHC class II molecule and CD8+ T cells to a MHC class Imolecule. MHC class I and MHC class II molecules have distinctdistributions among cells that reflect the different effector functionsof the T cells that recognize them. MHC class I molecules presentpeptides of cytosolic and nuclear origin e.g. from pathogens, commonlyviruses, to CD8+ T cells, which differentiate into cytotoxic T cellsthat are specialized to kill any cell that they specifically recognize.Almost all cells express MHC class I molecules, although the level ofconstitutive expression varies from one cell type to the next. But notonly pathogenic peptides from viruses are presented by MHC class Imolecules, also self-antigens like tumor antigens are presented by them.MHC class I molecules bind peptides from proteins degraded in thecytosol and transported in the endoplasmic reticulum. The CD8+ T cellsthat recognize MHC class I:peptide complexes at the surface of infectedcells are specialized to kill any cells displaying foreign peptides andso rid the body of cells infected with viruses and other cytosolicpathogens. The main function of CD4+ T cells (CD4+ helper T cells) thatrecognize MHC class II molecules is to activate other effector cells ofthe immune system. Thus MHC class II molecules are normally found on Blymphocytes, dendritic cells, and macrophages, cells that participate inimmune responses, but not on other tissue cells. Macrophages, forexample, are activated to kill the intravesicular pathogens theyharbour, and B cells to secrete immunoglobulins against foreignmolecules. MHC class II molecules are prevented from binding to peptidesin the endoplasmic reticulum and thus MHC class II molecules bindpeptides from proteins which are degraded in endosomes. They can capturepeptides from pathogens that have entered the vesicular system ofmacrophages, or from antigens internalized by immature dendritic cellsor the immunoglobulin receptors of B cells. Pathogens that accumulate inlarge numbers inside macrophage and dendritic cell vesicles tend tostimulate the differentiation of Th1 cells, whereas extracellularantigens tend to stimulate the production of Th2 cells. Th1 cellsactivate the microbicidal properties of macrophages and induce B cellsto make IgG antibodies that are very effective of opsonisingextracellular pathogens for ingestion by phagocytic cells, whereas Th2cells initiate the humoral response by activating naïve B cells tosecrete IgM, and induce the production of weakly opsonising antibodiessuch as IgG1 and IgG3 (mouse) and IgG2 and IgG4 (human) as well as IgAand IgE (mouse and human).

Epitope (also called “antigen determinant”): T cell epitopes maycomprise fragments preferably having a length of about 6 to about 20 oreven more amino acids, e.g. fragments as processed and presented by MHCclass I molecules, preferably having a length of about 8 to about 10amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), orfragments as processed and presented by MHC class II molecules,preferably having a length of about 13 or more amino acids, e.g. 13, 14,15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragmentsmay be selected from any part of the amino acid sequence. Thesefragments are typically recognized by T cells in form of a complexconsisting of the peptide fragment and an MHC molecule. B cell epitopesare typically fragments located on the outer surface of (native) proteinor peptide antigens.

Vaccine: A vaccine is typically understood to be a prophylactic ortherapeutic material providing at least one antigen or antigenicfunction. The antigen or antigenic function may stimulate the body'sadaptive immune system to provide an adaptive immune response.

Antigen-providing mRNA: An antigen-providing mRNA may typically be anmRNA, having at least one open reading frame that can be translated by acell or an organism provided with that mRNA. The product of thistranslation is a peptide or protein that may act as an antigen,preferably as an immunogen. The product may also be a fusion proteincomposed of more than one immunogen, e.g. a fusion protein that consistof two or more epitopes, peptides or proteins, wherein the epitopes,peptides or proteins may be linked by linker sequences.

Bi-/multicistronic mRNA: An bi-/multicistronic mRNA typically may havetwo (bicistronic) or more (multicistronic) coding sequences (cds) (alsooften referred to as open reading frames (ORF)). A coding sequence/anopen reading frame in this context is a sequence of several nucleotidetriplets (codons) that can be translated into a peptide or protein.Translation of such an mRNA yields two (bicistronic) or more(multicistronic) distinct translation products (provided the codingsequences/ORFs are not identical). For expression in eukaryotes suchmRNAs may for example comprise an internal ribosomal entry site (IRES)sequence.

5′-CAP-Structure: A 5′-CAP is typically a modified nucleotide (CAPanalogue), particularly a guanine nucleotide, added to the 5′ end of anmRNA molecule. Preferably, the 5′-CAP is added using a5′-5′-triphosphate linkage (also named m7GpppN). Further examples of5′-CAP structures include glyceryl, inverted deoxy abasic residue(moiety), 4′,5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl)nucleotide, 4′-thio nucleotide, carbocyclic nucleotide,1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modifiedbase nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4′-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety,3′-3′-inverted abasic moiety, 3′-2′-inverted nucleotide moiety,3′-2′-inverted abasic moiety, 1,4-butanediol phosphate,3′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,3′phosphorothioate, phosphorodithioate, or bridging or non-bridgingmethylphosphonate moiety. These modified 5′-CAP structures may be usedin the context of the present invention to modify the mRNA sequence ofthe inventive composition. Further modified 5′-CAP structures which maybe used in the context of the present invention are CAP1 (additionalmethylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2(additional methylation of the ribose of the 2^(nd) nucleotidedownstream of the m7GpppN), CAP3 (additional methylation of the riboseof the 3^(rd) nucleotide downstream of the m7GpppN), CAP4 (additionalmethylation of the ribose of the 4^(th) nucleotide downstream of them7GpppN), ARCA (anti-reverse CAP analogue), modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

In the context of the present invention, a 5′ cap structure may also beformed in chemical RNA synthesis or RNA in vitro transcription(co-transcriptional capping) using cap analogues, or a cap structure maybe formed in vitro using capping enzymes (e.g., commercially availablecapping kits)

Cap analogue: A cap analogue refers to a non-polymerizable di-nucleotidethat has cap functionality in that it facilitates translation orlocalization, and/or prevents degradation of the RNA molecule whenincorporated at the 5′ end of the RNA molecule. Non-polymerizable meansthat the cap analogue will be incorporated only at the 5′terminusbecause it does not have a 5′ triphosphate and therefore cannot beextended in the 3′ direction by a template-dependent RNA polymerase.

Cap analogues include, but are not limited to, a chemical structureselected from the group consisting of m7GpppG, m7GpppA, m7GpppC;unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue(e.g., m2,7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG),dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or antireverse cap analogues (e.g., ARCA; m7,2′OmeGpppG, m7,2′dGpppG,m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives)(Stepinski et al., 2001. RNA 7(10):1486-95).

Further cap analogues have been described previously (U.S. Pat. No.7,074,596, WO 2008/016473, WO 2008/157688, WO 2009/149253, WO2011/015347, and WO 2013/059475). The synthesis ofN⁷-(4-chlorophenoxyethyl) substituted dinucleotide cap analogues hasbeen described recently (Kore et al. (2013) Bioorg. Med. Chem. 21(15):4570-4).

Fragments of proteins: “Fragments” of proteins or peptides in thecontext of the present invention may, typically, comprise a sequence ofa protein or peptide as defined herein, which is, with regard to itsamino acid sequence (or its encoded nucleic acid molecule), N-terminallyand/or C-terminally truncated compared to the amino acid sequence of theoriginal (native) protein (or its encoded nucleic acid molecule). Suchtruncation may thus occur either on the amino acid level orcorrespondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid molecule of such a protein or peptide. Inthe context of antigens such fragment may have a length of about 6 toabout 20 or even more amino acids, e.g. fragments as processed andpresented by MHC class I molecules, preferably having a length of about8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12amino acids), or fragments as processed and presented by MHC class IImolecules, preferably having a length of about 13 or more amino acids,e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, whereinthese fragments may be selected from any part of the amino acidsequence. These fragments are typically recognized by T-cells in form ofa complex consisting of the peptide fragment and an MHC molecule, i.e.the fragments are typically not recognized in their native form.Fragments of proteins or peptides (e.g. in the context of antigens) maycomprise at least one epitope of those proteins or peptides. Furthermorealso domains of a protein, like the extracellular domain, theintracellular domain or the transmembrane domain and shortened ortruncated versions of a protein may be understood to comprise a fragmentof a protein. Preferably, a fragment of a protein comprises a functionalfragment of the protein, which means that the fragment exerts the sameeffect or functionality as the whole protein it is derived from.

Variants of proteins: “Variants” of proteins or peptides as defined inthe context of the present invention may be generated, having an aminoacid sequence which differs from the original sequence in one or moremutation(s), such as one or more substituted, inserted and/or deletedamino acid(s). Preferably, these fragments and/or variants have the samebiological function or specific activity compared to the full-lengthnative protein, e.g. its specific antigenic property. “Variants” ofproteins or peptides as defined in the context of the present inventionmay comprise conservative amino acid substitution(s) compared to theirnative, i.e. non-mutated physiological, sequence. Those amino acidsequences as well as their encoding nucleotide sequences in particularfall under the term variants as defined herein. Substitutions in whichamino acids, which originate from the same class, are exchanged for oneanother are called conservative substitutions. In particular, these areamino acids having aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or amino acids,the side chains of which can enter into hydrogen bridges, e.g. sidechains which have a hydroxyl function. This means that e.g. an aminoacid having a polar side chain is replaced by another amino acid havinga likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam).

A “variant” of a protein or peptide may have at least 70%, 75%, 80%,85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20,30, 50, 75 or 100 amino acids of such protein or peptide. Furthermore,variants of proteins or peptides as defined herein, which may be encodedby a nucleic acid molecule, may also comprise those sequences, whereinnucleotides of the encoding nucleic acid sequence are exchangedaccording to the degeneration of the genetic code, without leading to analteration of the respective amino acid sequence of the protein orpeptide, i.e. the amino acid sequence or at least part thereof may notdiffer from the original sequence within the above meaning. Preferably,a variant of a protein comprises a functional variant of the protein,which means that the variant exerts the same effect or functionality asthe protein it is derived from.

Identity of a sequence: In order to determine the percentage to whichtwo sequences are identical, e.g. nucleic acid sequences or amino acidsequences as defined herein, preferably the amino acid sequences encodedby a nucleic acid sequence of the polymeric carrier as defined herein orthe amino acid sequences themselves, the sequences can be aligned inorder to be subsequently compared to one another. Therefore, e.g. aposition of a first sequence may be compared with the correspondingposition of the second sequence. If a position in the first sequence isoccupied by the same component (residue) as is the case at a position inthe second sequence, the two sequences are identical at this position.If this is not the case, the sequences differ at this position. Ifinsertions occur in the second sequence in comparison to the firstsequence, gaps can be inserted into the first sequence to allow afurther alignment. If deletions occur in the second sequence incomparison to the first sequence, gaps can be inserted into the secondsequence to allow a further alignment. The percentage to which twosequences are identical is then a function of the number of identicalpositions divided by the total number of positions including thosepositions which are only occupied in one sequence. The percentage towhich two sequences are identical can be determined using a mathematicalalgorithm. A preferred, but not limiting, example of a mathematicalalgorithm which can be used is the algorithm of Karlin et al. (1993),PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res.,25:3389-3402. Such an algorithm is integrated in the BLAST program.Sequences which are identical to the sequences of the present inventionto a certain extent can be identified by this program.

Monocistronic mRNA: A monocistronic mRNA may typically be an mRNA, thatcomprises only one coding sequence (open reading frame). A codingsequence/open reading frame in this context is a sequence of severalnucleotide triplets (codons) that can be translated into a peptide orprotein.

Nucleic acid: The term nucleic acid means any DNA or RNA molecule and isused synonymous with polynucleotide. Wherever herein reference is madeto a nucleic acid or nucleic acid sequence encoding a particular proteinand/or peptide, said nucleic acid or nucleic acid sequence,respectively, preferably also comprises regulatory sequences allowing ina suitable host, e.g. a human being, its expression, i.e. transcriptionand/or translation of the nucleic acid sequence encoding the particularprotein or peptide.

Peptide: A peptide is a polymer of amino acid monomers. Usually themonomers are linked by peptide bonds. The term “peptide” does not limitthe length of the polymer chain of amino acids. In some embodiments ofthe present invention a peptide may for example contain less than 50monomer units. Longer peptides are also called polypeptides, typicallyhaving 50 to 600 monomeric units, more specifically 50 to 300 monomericunits.

Pharmaceutically effective amount: A pharmaceutically effective amountin the context of the invention is typically understood to be an amountthat is sufficient to induce an immune response or to trigger thedesired therapeutical effect.

Protein: A protein typically consists of one or more peptides and/orpolypeptides folded into 3-dimensional form, facilitating a biologicalfunction.

Poly(C) sequence: A poly(C) sequence is typically a long sequence ofcytosine nucleotides, typically about 10 to about 200 cytosinenucleotides, preferably about 10 to about 100 cytosine nucleotides, morepreferably about 10 to about 70 cytosine nucleotides or even more,preferably about 20 to about 50, or even about 20 to about 30 cytosinenucleotides. A poly(C) sequence may preferably be located 3′ of thecoding region comprised by a nucleic acid.

Poly(A) tail: A poly(A) tail also called “3′-poly(A) tail” or “Poly(A)sequence” is typically a long homopolymeric sequence of adenosinenucleotides of up to about 400 adenosine nucleotides, e.g. from about 25to about 400, preferably from about 50 to about 400, more preferablyfrom about 50 to about 300, even more preferably from about 50 to about250, most preferably from about 60 to about 250 adenosine nucleotides,added to the 3′ end of an mRNA. In the context of the present invention,the poly(A) tail of an mRNA is preferably derived from a DNA template byRNA in vitro transcription. Alternatively, the poly(A) sequence may alsobe obtained in vitro by common methods of chemical synthesis withoutbeing necessarily transcribed from a DNA-progenitor. Moreover, poly(A)sequences, or poly(A) tails may be generated by enzymaticpolyadenylation of the RNA.

Stabilized nucleic acid: A stabilized nucleic acid, typically, exhibitsa modification increasing resistance to in vivo degradation (e.g.degradation by an exo- or endo-nuclease) and/or ex vivo degradation(e.g. by the manufacturing process prior to vaccine administration, e.g.in the course of the preparation of the vaccine solution to beadministered). Stabilization of RNA can, e.g., be achieved by providinga 5′-CAP-Structure, a poly(A) tail, or any other UTR-modification. Itcan also be achieved by backbone-modification or modification of theG/C-content or the C-content of the nucleic acid. Various other methodsare known in the art and conceivable in the context of the invention.

Carrier/polymeric carrier: A carrier in the context of the invention maytypically be a compound that facilitates transport and/or complexationof another compound. Said carrier may form a complex with said othercompound. A polymeric carrier is a carrier that is formed of a polymer.

Cationic component: The term “cationic component” typically refers to acharged molecule, which is positively charged (cation) at a pH value oftypically about 1 to 9, preferably of a pH value of or below 9 (e.g. 5to 9), of or below 8 (e.g. 5 to 8), of or below 7 (e.g. 5 to 7), mostpreferably at physiological pH values, e.g. about 7.3 to 7.4.Accordingly, a cationic peptide, protein or polymer according to thepresent invention is positively charged under physiological conditions,particularly under physiological salt conditions of the cell in vivo. Acationic peptide or protein preferably contains a larger number ofcationic amino acids, e.g. a larger number of Arg, His, Lys or Orn thanother amino acid residues (in particular more cationic amino acids thananionic amino acid residues like Asp or Glu) or contains blockspredominantly formed by cationic amino acid residues. The definition“cationic” may also refer to “polycationic” components.

Vehicle: A vehicle is an agent, e.g. a carrier, that may typically beused within a pharmaceutical composition or vaccine for facilitatingadministering of the components of the pharmaceutical composition orvaccine to an individual. 3′-untranslated region (3′-UTR): A 3′-UTR istypically the part of an mRNA which is located between the proteincoding region (i.e. the open reading frame) and the poly(A) sequence ofthe mRNA. A 3′-UTR of the mRNA is not translated into an amino acidsequence. The 3′-UTR sequence is generally encoded by the gene which istranscribed into the respective mRNA during the gene expression process.The genomic sequence is first transcribed into pre-mature mRNA, whichcomprises optional introns. The pre-mature mRNA is then furtherprocessed into mature mRNA in a maturation process. This maturationprocess comprises the steps of 5′-capping, splicing the pre-mature mRNAto excise optional introns and modifications of the 3′-end, such aspolyadenylation of the 3′-end of the pre-mature mRNA and optional endo-or exonuclease cleavages etc. In the context of the present invention, a3′-UTR corresponds to the sequence of a mature mRNA which is located 3′to the stop codon of the protein coding region, preferably immediately3′ to the stop codon of the protein coding region, and which extends tothe 5′-side of the poly(A) sequence, preferably to the nucleotideimmediately 5′ to the poly(A) sequence. The term “corresponds to” meansthat the 3′-UTR sequence may be an RNA sequence, such as in the mRNAsequence used for defining the 3′-UTR sequence, or a DNA sequence whichcorresponds to such RNA sequence. In the context of the presentinvention, the term “a 3′-UTR of a gene”, such as “a 3′-UTR of analbumin gene”, is the sequence which corresponds to the 3′-UTR of themature mRNA derived from this gene, i.e. the mRNA obtained bytranscription of the gene and maturation of the pre-mature mRNA. Theterm “3′-UTR of a gene” encompasses the DNA sequence and the RNAsequence of the 3′-UTR.

5′-untranslated region (5′-UTR): A 5′-UTR is typically understood to bea particular section of messenger RNA (mRNA). It is located 5′ of theopen reading frame of the mRNA. Typically, the 5′-UTR starts with thetranscriptional start site and ends one nucleotide before the startcodon of the open reading frame. The 5′-UTR may comprise elements forcontrolling gene expression, also called regulatory elements. Suchregulatory elements may be, for example, ribosomal binding sites or a5′-Terminal Oligopyrimidine Tract. The 5′-UTR may beposttranscriptionally modified, for example by addition of a 5′-CAP. Inthe context of the present invention, a 5′UTR corresponds to thesequence of a mature mRNA which is located between the 5′-CAP and thestart codon. Preferably, the 5′-UTR corresponds to the sequence whichextends from a nucleotide located 3′ to the 5′-CAP, preferably from thenucleotide located immediately 3′ to the 5′-CAP, to a nucleotide located5′ to the start codon of the protein coding region, preferably to thenucleotide located immediately 5′ to the start codon of the proteincoding region. The nucleotide located immediately 3′ to the 5′-CAP of amature mRNA typically corresponds to the transcriptional start site. Theterm “corresponds to” means that the 5′-UTR sequence may be an RNAsequence, such as in the mRNA sequence used for defining the 5′-UTRsequence, or a DNA sequence which corresponds to such RNA sequence. Inthe context of the present invention, the term “a 5′-UTR of a gene”,such as “a 5′-UTR of a TOP gene”, is the sequence which corresponds tothe 5′-UTR of the mature mRNA derived from this gene, i.e. the mRNAobtained by transcription of the gene and maturation of the pre-maturemRNA. The term “5′-UTR of a gene” encompasses the DNA sequence and theRNA sequence of the 5′-UTR.

5′ Terminal Oligopyrimidine Tract (TOP): The 5′ terminal oligopyrimidinetract (TOP) is typically a stretch of pyrimidine nucleotides located atthe 5′ terminal region of a nucleic acid molecule, such as the 5′terminal region of certain mRNA molecules or the 5′ terminal region of afunctional entity, e.g. the transcribed region, of certain genes. Thesequence starts with a cytidine, which usually corresponds to thetranscriptional start site, and is followed by a stretch of usuallyabout 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidinestretch and thus the 5′ TOP ends one nucleotide 5′ to the first purinenucleotide located downstream of the TOP. mRNA that contains a 5′terminal oligopyrimidine tract is often referred to as TOP mRNA.Accordingly, genes that provide such messenger RNAs are referred to asTOP genes. TOP sequences have, for example, been found in genes andmRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, a TOP motif is anucleic acid sequence which corresponds to a 5′ TOP as defined above.Thus, a TOP motif in the context of the present invention is preferablya stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides,preferably at least 4 pyrimidine nucleotides, preferably at least 5pyrimidine nucleotides, more preferably at least 6 nucleotides, morepreferably at least 7 nucleotides, most preferably at least 8 pyrimidinenucleotides, wherein the stretch of pyrimidine nucleotides preferablystarts at its 5′ end with a cytosine nucleotide. In TOP genes and TOPmRNAs, the TOP-motif preferably starts at its 5′ end with thetranscriptional start site and ends one nucleotide 5′ to the firstpurine residue in said gene or mRNA. A TOP motif in the sense of thepresent invention is preferably located at the 5′end of a sequence whichrepresents a 5′-UTR or at the 5′ end of a sequence which codes for a5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotidesis called “TOP motif” in the sense of the present invention if thisstretch is located at the 5′ end of a respective sequence, such as theinventive mRNA, the 5′-UTR element of the inventive mRNA, or the nucleicacid sequence which is derived from the 5′-UTR of a TOP gene asdescribed herein. In other words, a stretch of 3 or more pyrimidinenucleotides which is not located at the 5′-end of a 5′-UTR or a 5′-UTRelement but anywhere within a 5′-UTR or a 5′-UTR element is preferablynot referred to as “TOP motif”.

TOP gene: TOP genes are typically characterised by the presence of a 5′terminal oligopyrimidine tract. Furthermore, most TOP genes arecharacterized by a growth-associated translational regulation. However,also TOP genes with a tissue specific translational regulation areknown. As defined above, the 5′-UTR of a TOP gene corresponds to thesequence of a 5′-UTR of a mature mRNA derived from a TOP gene, whichpreferably extends from the nucleotide located 3′ to the 5′-CAP to thenucleotide located 5′ to the start codon. A 5′-UTR of a TOP genetypically does not comprise any start codons, preferably no upstreamAUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstreamAUGs and upstream open reading frames are typically understood to beAUGs and open reading frames that occur 5′ of the start codon (AUG) ofthe open reading frame that should be translated. The 5′-UTRs of TOPgenes are generally rather short. The lengths of 5′-UTRs of TOP genesmay vary between 20 nucleotides up to 500 nucleotides, and are typicallyless than about 200 nucleotides, preferably less than about 150nucleotides, more preferably less than about 100 nucleotides. Exemplary5′-UTRs of TOP genes in the sense of the present invention are thenucleic acid sequences extending from the nucleotide at position 5 tothe nucleotide located immediately 5′ to the start codon (e.g. the ATG)in the sequences according to SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQID NO. 1421 and SEQ ID NO. 1422 of the international patent applicationWO2013/143700 or homologs or variants thereof, whose disclosure isincorporated herewith by reference. In this context a particularlypreferred fragment of a 5′UTR of a TOP gene is a 5′-UTR of a TOP genelacking the 5′ TOP motif. The term ‘5′UTR of a TOP gene’ preferablyrefers to the 5′-UTR of a naturally occurring TOP gene.

Chemical synthesis of RNA: Chemical synthesis of relatively shortfragments of oligonucleotides with defined chemical structure provides arapid and inexpensive access to custom-made oligonucleotides of anydesired sequence. Whereas enzymes synthesize DNA and RNA only in the 5′to 3′ direction, chemical oligonucleotide synthesis does not have thislimitation, although it is most often carried out in the opposite, i.e.the 3′ to 5′ direction. Currently, the process is implemented assolid-phase synthesis using the phosphoramidite method andphosphoramidite building blocks derived from protected nucleosides (A,C, G, and U), or chemically modified nucleosides.

To obtain the desired oligonucleotide, the building blocks aresequentially coupled to the growing oligonucleotide chain on a solidphase in the order required by the sequence of the product in a fullyautomated process. Upon the completion of the chain assembly, theproduct is released from the solid phase to the solution, deprotected,and collected. The occurrence of side reactions sets practical limitsfor the length of synthetic oligonucleotides (up to about 200 nucleotideresidues), because the number of errors increases with the length of theoligonucleotide being synthesized. Products are often isolated by HPLCto obtain the desired oligonucleotides in high purity.

Chemically synthesized oligonucleotides find a variety of applicationsin molecular biology and medicine. They are most commonly used asantisense oligonucleotides, small interfering RNA, primers for DNAsequencing and amplification, probes for detecting complementary DNA orRNA via molecular hybridization, tools for the targeted introduction ofmutations and restriction sites, and for the synthesis of artificialgenes.

RNA In vitro transcription: The terms “RNA in vitro transcription” or“in vitro transcription” relate to a process wherein RNA is synthesizedin a cell-free system (in vitro). DNA, particularly plasmid DNA, is usedas template for the generation of RNA transcripts. RNA may be obtainedby DNA-dependent in vitro transcription of an appropriate DNA template,which according to the present invention is preferably a linearizedplasmid DNA template. The promoter for controlling in vitrotranscription can be any promoter for any DNA-dependent RNA polymerase.Particular examples of DNA-dependent RNA polymerases are the T7, T3, andSP6 RNA polymerases. A DNA template for in vitro RNA transcription maybe obtained by cloning of a nucleic acid, in particular cDNAcorresponding to the respective RNA to be in vitro transcribed, andintroducing it into an appropriate vector for in vitro transcription,for example into plasmid DNA. In a preferred embodiment of the presentinvention the DNA template is linearized with a suitable restrictionenzyme, before it is transcribed in vitro. The cDNA may be obtained byreverse transcription of mRNA or chemical synthesis. Moreover, the DNAtemplate for in vitro RNA synthesis may also be obtained by genesynthesis.

Methods for in vitro transcription are known in the art (see, e.g.,Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al.(2013) Methods Enzymol. 530:101-14). Reagents used in said methodtypically include:

1) a linearized DNA template with a promoter sequence that has a highbinding affinity for its respective RNA polymerase such asbacteriophage-encoded RNA polymerases;

2) ribonucleoside triphosphates (NTPs) for the four bases (adenine,cytosine, guanine and uracil);

3) optionally a cap analogue as defined above (e.g. m7G(5′)ppp(5′)G(m7G));

4) a DNA-dependent RNA polymerase capable of binding to the promotersequence within the linearized DNA template (e.g. T7, T3 or SP6 RNApolymerase);

5) optionally a ribonuclease (RNase) inhibitor to inactivate anycontaminating RNase;

6) optionally a pyrophosphatase to degrade pyrophosphate, which mayinhibit transcription;

7) MgCl₂, which supplies Mg²⁺ ions as a co-factor for the polymerase;

8) a buffer to maintain a suitable pH value, which can also containantioxidants (e.g. DTT), and/or polyamines such as spermidine at optimalconcentrations.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotide monomers.These nucleotides are usually adenosine monophosphate (AMP), uridinemonophosphate (UMP), guanosine monophosphate (GMP) and cytidinemonophosphate (CMP) monomers or analogues thereof, which are connectedto each other along a so-called backbone. The backbone is formed byphosphodiester bonds between the sugar, i.e. ribose, of a first and aphosphate moiety of a second, adjacent monomer. The specific order ofthe monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the RNA sequence. Usually RNA may beobtainable by transcription of a DNA sequence, e.g., inside a cell. Ineukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. In vivo, transcription of DNA usuallyresults in the so-called premature RNA (also called pre-mRNA, precursormRNA or heterogeneous nuclear RNA) which has to be processed intoso-called messenger RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap,optionally a 5′UTR, an open reading frame, optionally a 3′UTR and apoly(A) tail.

In addition to messenger RNA, several non-coding types of RNA existwhich may be involved in regulation of transcription and/or translation,and immunostimulation. Within the present invention the term “RNA”further encompasses any type of single stranded (ssRNA) or doublestranded RNA (d5RNA) molecule known in the art, such as viral RNA,retroviral RNA and replicon RNA, small interfering RNA (siRNA),antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers,riboswitches, immunostimulating/immunostimulatory RNA, transfer RNA(tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolarRNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

Fragment of a nucleic acid sequence, particularly an RNA: A fragment ofa nucleic acid sequence consists of a continuous stretch of nucleotidescorresponding to a continuous stretch of nucleotides in the full-lengthnucleic acid sequence which is the basis for the nucleic acid sequenceof the fragment, which represents at least 20%, preferably at least 30%,more preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, and most preferably at least 90% of thefull-length nucleic acid sequence. Such a fragment, in the sense of thepresent invention, is preferably a functional fragment of thefull-length nucleic acid sequence.

Variant of a nucleic acid sequence, particularly anRNA: A variant of anucleic acid sequence refers to a variant of nucleic acid sequenceswhich forms the basis of a nucleic acid sequence. For example, a variantnucleic acid sequence may exhibit one or more nucleotide deletions,insertions, additions and/or substitutions compared to the nucleic acidsequence from which the variant is derived. Preferably, a variant of anucleic acid sequence is at least 40%, preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, even morepreferably at least 80%, even more preferably at least 90%, mostpreferably at least 95% identical to the nucleic acid sequence thevariant is derived from. Preferably, the variant is a functionalvariant. A “variant” of a nucleic acid sequence may have at least 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretchof 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid sequence.

Intratumoral administration/application: The term “intratumoraladministration/application” refers to the direct delivery of apharmaceutical composition into or adjacent to a tumor or cancer and/orimmediate vicinity of a tumor or cancer. Multiple injections intoseparate regions of the tumor or cancer are also included. Furthermore,intratumoral administration/application includes delivery of apharmaceutical composition into one or more metastases.

Methods for intratumoral delivery of drugs are known in the art(Brincker, 1993. Crit. Rev. Oncol. Hematol. 15(2):91-8; Celikoglu etal., 2008. Cancer Therapy 6, 545-552). For example, the pharmaceuticalcomposition can be administered by conventional needle injection,needle-free jet injection or electroporation or combinations thereofinto the tumor or cancer tissue. The pharmaceutical composition can beinjected directly into the tumor or cancer (tissue) with great precisionusing computer tomograpy, ultrasound, gamma camera imaging, positronemission tomography, or magnetic resonance tumor imaging. Furtherprocedures are selected from the group including, but not limited to,direct intratumoral injection by endoscopy, bronchoscopy, cystoscopy,colonoscopy, laparoscope and catheterization.

Decoy receptors: Decoy receptors recognize certain growth factors orcytokines with high affinity and specificity, but are structurallyincapable of signaling or presenting the agonist to signaling receptorcomplexes. They act as a molecular trap for the agonist and forsignaling receptor components. A decoy receptor, or sink receptor, is areceptor that binds a ligand, inhibiting it from binding to its normalreceptor. For instance, the receptor VEGFR-1 can prevent vascularendothelial growth factor (VEGF) from binding to the VEGFR-2.

Dominant negative receptors: Dominant negative receptors are variants ofthe particular receptor comprising dominant-negative (DN) mutations asleading to mutant polypeptides that disrupt the activity of thewild-type receptor when overexpressed.

DETAILED DESCRIPTION OF THE INVENTION

The RNA containing composition according to the invention comprises atleast one RNA and is particularly provided for use in the treatment orprophylaxis of tumor and/or cancer diseases, wherein the RNA containingcomposition is preferably applied/administered intratumorally. It isespecially preferred that the RNA containing composition is injecteddirectly into tumor tissue. Alternatively, it is especially preferredthat the RNA containing composition is injected adjacent to or in closeproximity to a tumor tissue and/or metastasis.

It has been found by the inventors that intratumoral applicationrespectively administration of the RNA containing composition accordingto the invention is capable of effectively treating tumor and/or cancerdiseases and related disorders. It has been shown that intratumoralapplication is surprisingly effective in decreasing tumor size. Moreoverthe application of the RNA containing composition according to theinvention was able to increase survival in animal models.

The at least one RNA of the RNA containing composition may be selectedfrom the group consisting of chemically modified or unmodified RNA,single-stranded or double-stranded RNA, coding or non-coding RNA, mRNA,oligoribonucleotide, viral RNA, retroviral RNA, replicon RNA, tRNA,rRNA, immunostimulatory RNA, microRNA, siRNA, small nuclear RNA (snRNA),small-hairpin (sh) RNA riboswitch, RNA aptamer, RNA decoy, antisenseRNA, a ribozyme, or any combination thereof. In specific embodiments theat least one RNA of the RNA containing composition is selected from acoding RNA or a non-coding RNA.

Coding RNA:

According to a preferred embodiment of the invention the at least oneRNA of the RNA containing composition comprises at least one codingregion encoding at least one peptide or protein. Preferably, the codingRNA is selected from the group consisting of mRNA, viral RNA, retroviralRNA, and replicon RNA.

In preferred embodiments of the invention the at least one RNA of theRNA containing composition codes for at least one cytokine and/or for atleast one chemokine and/or for at least one suicide gene product, and/orat least one immunogenic protein or peptide and/or for at least one celldeath/apoptosis inducer and/or for at least one angiogenesis inhibitorand/or for at least one heat shock protein and/or for at least one tumorantigen and/or for at least one β-catenin inhibitor and/or for at leastone activator of the STING (stimulator of interferon genes) pathwayand/or at least one checkpoint modulator and/or at least one antibody,and/or at least one dominant negative receptor, and/or at least onedecoy receptor, and/or at least one inhibitor of myeloid derivedsuppressor cells (MDSCs), and/or at least one IDO pathway inhibitor,and/or at least one protein or peptide that bind inhibitors ofapoptosis, or fragments or variants thereof as will be outlined in moredetail below.

1. Cytokines

In a preferred embodiment of the inventive RNA containing compositionthe RNA comprises at least one coding region that codes for at least onecytokine, or a fragment or variant thereof.

Preferably the cytokine is an interleukin (IL). One or more interleukinsmay be chosen e.g. from the following list: IL-1α, IL-1β, IL-1ra(antagonist), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10;IL-11, IL-12, IL-13, IL14, IL-15, IL-16, IL-17A, IL-17B, EL-17C, IL-17D,IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, IL-27, IL-28A/B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35.Moreover the cytokine may be one or more cytokines chosen from the TNFfamily, e.g. chosen from the following list: TNF, especially TNFα, LTα,LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L (CD154), FASL,CD27L, CD30L, 4-1BBL, TRAIL, RANK ligand. Further examples of preferredcytokines may be chosen from the following list: FLT3 ligand, G-CSF,GM-CSF, IFNα/β/ω, IFNγ, LIF, M-CSF, MIF, OSM, Stem Cell Factor, TGFβ1,TGFβ2, TGFβ3, TSLP ligand.

Particularly preferred are cytokines chosen from the following list:IL-12, IL-15, IL-2, IFNγ, TNFα, IL-18, IFNα, IL-1β, IL-32, IL-7, IL-21,IL-8, GM-CSF.

In an especially preferred embodiment of the invention the RNA of theinventive composition codes for Interleukin-12 or CD40L. It has beenshown by the inventors, that mRNA coding for this cytokines isespecially effective when applied in the inventive approach.Particularly preferred are RNA sequences according to SEQ ID Nos. 1, 3,4194, 4195, 4196, 4197, 4198, 4199, 4200 encoding IL-12. Furthermore RNAsequences according to SEQ ID Nos. 3898, 3899, 3900, 3901, 3902, 3903,3904, 10073, encoding CD40L are particularly preferred.

According to preferred embodiments in the context of the presentinvention cytokines may be selected from any cytokine selected from thegroup consisting of 4-1BBL; Apo2L/TRAIL; APRIL; BAFF; CD27L; CD30L;CD40L_(CD154); CXCL8; EL-17C; FasL; FLT3_ligand; G-CSF; GITRL; GM-CSF;IFNalpha; IFNB; IFNG; IFNomega; IL-1_alpha; IL-1_beta; IL-10; IL-11;IL-12; IL-12A; IL-13; IL-14; IL-15; IL-16; IL-17A; IL-17B; IL-17D;IL-17F; IL-18; IL-19; IL-1ra_(antagonist); IL-2; IL-20; IL-21; IL-22;IL-23; IL-24; IL-25; IL-26; IL-27A; IL-27B; IL-28A; IL-28B; IL-29; IL-3;IL-31; IL-32; IL-33; IL-37; IL-4; IL-5; IL-6; IL-7; IL-9; LIF; LIGHT;LTalpha; LTbeta; M-CSF; MIF; OSM; OX40L; RANK_ligand; Stem_Cell_Factor;TGFbeta1; TGFbeta2; TGFbeta3; TL1A; TNF; TWEAK, preferably as disclosedin Table 1. Particularly preferred in this context are the RNA sequencesencoding a cytokine according to Table 1.

TABLE 1 Cytokines: RNA Protein Sequence Sequence wild type Optimized SEQID SEQ ID RNA Sequence Gene Name Protein Accession No. NO: NO: SEQ IDNO: 4-1BBL UniProtKB: P41273 3849 3850 3851, 3852, 3853, 3854, 3855,3856 APRIL UniProtKB: O75888 3857 3858 3859, 3860, 3861, 3862, 3863,3864 BAFF UniProtKB: Q5H8V1 3865 3866 3867, 3868, 3869, 3870, 3871, 3872BAFF UniProtKB: Q9Y275 3873 3874 3875, 3876, 3877, 3878, 3879, 3880CD27L UniProtKB: P32970 3881 3882 3883, 3884, 3885, 3886, 3887, 3888CD30L UniProtKB: P32971 3889 3890 3891, 3892, 3893, 3894, 3895, 3896CD40L_(CD154) UniProtKB: P29965 3897 3898 3899, 3900, 3901, 3902, 3903,3904 EL-17C UniProtKB: Q9P0M4 3905 3906 3907, 3908, 3909, 3910, 3911,3912 FLT3_ligand Genbank: AAA90950.1 3913 3914 3915, 3916, 3917, 3918,3919, 3920 FLT3_ligand UniProtKB: P49771 3921 3922 3923, 3924, 3925,3926, 3927, 3928 G-CSF UniProtKB: P09919 3929 3930 3931, 3932, 3933,3934, 3935, 3936 GITRL UniProtKB: Q9UNG2 3937 3938 3939, 3940, 3941,3942, 3943, 3944 GM-CSF UniProtKB: P04141 3945 3946 3947, 3948, 3949,3950, 3951, 3952 IFNalpha UniProtKB: G9JKF1 3953 3954 3955, 3956, 3957,3958, 3959, 3960 IFNalpha UniProtKB: P01562 3961 3962 3963, 3964, 3965,3966, 3967, 3968 IFNalpha UniProtKB: P01563 3969 3970 3971, 3972, 3973,3974, 3975, 3976 IFNalpha UniProtKB: P01566 3977 3978 3979, 3980, 3981,3982, 3983, 3984 IFNalpha UniProtKB: P01567 3985 3986 3987, 3988, 3989,3990, 3991, 3992 IFNalpha UniProtKB: P01568 3993 3994 3995, 3996, 3997,3998, 3999, 4000 IFNalpha UniProtKB: P01569 4001 4002 4003, 4004, 4005,4006, 4007, 4008 IFNalpha UniProtKB: P01570 4009 4010 4011, 4012, 4013,4014, 4015, 4016 IFNalpha UniProtKB: P01571 4017 4018 4019, 4020, 4021,4022, 4023, 4024 IFNalpha UniProtKB: P05013 4025 4026 4027, 4028, 4029,4030, 4031, 4032 IFNalpha UniProtKB: P05014 4033 4034 4035, 4036, 4037,4038, 4039, 4040 IFNalpha UniProtKB: P05015 4041 4042 4043, 4044, 4045,4046, 4047, 4048 IFNalpha UniProtKB: P32881 4049 4050 4051, 4052, 4053,4054, 4055, 4056 IFNalpha UniProtKB: Q14618 4057 4058 4059, 4060, 4061,4062, 4063, 4064 IFNalpha UniProtKB: Q86UP4 4065 4066 4067, 4068, 4069,4070, 4071, 4072 IFNB UniProtKB: P01574 4073 4074 4075, 4076, 4077,4078, 4079, 4080 IFNB UniProtKB: Q15943 4081 4082 4083, 4084, 4085,4086, 4087, 4088 IFNG UniProtKB: P01579 4089 4090 4091, 4092, 4093,4094, 4095, 4096 IFNG UniProtKB: Q14609 4097 4098 4099, 4100, 4101,4102, 4103, 4104 IFNG UniProtKB: Q14610 4105 4106 4107, 4108, 4109,4110, 4111, 4112 IFNG UniProtKB: Q14611 4113 4114 4115, 4116, 4117,4118, 4119, 4120 IFNG UniProtKB: Q14612 4121 4122 4123, 4124, 4125,4126, 4127, 4128 IFNG UniProtKB: Q14613 4129 4130 4131, 4132, 4133,4134, 4135, 4136 IFNG UniProtKB: Q14614 4137 4138 4139, 4140, 4141,4142, 4143, 4144 IFNG UniProtKB: Q14615 4145 4146 4147, 4148, 4149,4150, 4151, 4152 IFNG UniProtKB: Q8NHY9 4153 4154 4155, 4156, 4157,4158, 4159, 4160 IFNomega UniProtKB: P05000 4161 4162 4163, 4164, 4165,4166, 4167, 4168 IL-10 UniProtKB: P22301 4169 4170 4171, 4172, 4173,4174, 4175, 4176 IL-11 UniProtKB: P20809 4177 4178 4179, 4180, 4181,4182, 4183, 4184 IL-12A UniProtKB: P29459 4185 4186 4187, 4188, 4189,4190, 4191, 4192 IL-12 UniProtKB: P29460 4193 4194 4195, 4196, 4197,4198, 4199, 4200 IL-13 UniProtKB: P35225 4201 4202 4203, 4204, 4205,4206, 4207, 4208 IL-14 UniProtKB: P40222 4209 4210 4211, 4212, 4213,4214, 4215, 4216 IL-15 UniProtKB: P40933 4217 4218 4219, 4220, 4221,4222, 4223, 4224 IL-16 UniProtKB: Q14005 4225 4226 4227, 4228, 4229,4230, 4231, 4232 IL-17A UniProtKB: Q16552 4233 4234 4235, 4236, 4237,4238, 4239, 4240 IL-17B UniProtKB: Q9NRM6 4241 4242 4243, 4244, 4245,4246, 4247, 4248 IL-17B UniProtKB: Q9UHF5 4249 4250 4251, 4252, 4253,4254, 4255, 4256 IL-17D UniProtKB: Q8TAD2 4257 4258 4259, 4260, 4261,4262, 4263, 4264 IL-17F UniProtKB: F1JZ09 4265 4266 4267, 4268, 4269,4270, 4271, 4272 IL-17F UniProtKB: Q96PD4 4273 4274 4275, 4276, 4277,4278, 4279, 4280 IL-18 UniProtKB: A0A024R3E0 4281 4282 4283, 4284, 4285,4286, 4287, 4288 IL-18 UniProtKB: B0YJ28 4289 4290 4291, 4292, 4293,4294, 4295, 4296 IL-18 UniProtKB: Q14116 4297 4298 4299, 4300, 4301,4302, 4303, 4304 IL-19 UniProtKB: Q9UHD0 4305 4306 4307, 4308, 4309,4310, 4311, 4312 IL-1_alpha UniProtKB: P01583 4313 4314 4315, 4316,4317, 4318, 4319, 4320 IL-1_beta UniProtKB: P01584 4321 4322 4323, 4324,4325, 4326, 4327, 4328 IL-1ra_(antagonist) UniProtKB: P18510-2 4329 43304331, 4332, 4333, 4334, 4335, 4336 IL-1ra_(antagonist) UniProtKB:P18510-3 4337 4338 4339, 4340, 4341, 4342, 4343, 4344IL-1ra_(antagonist) UniProtKB: P18510 4345 4346 4347, 4348, 4349, 4350,4351, 4352 IL-20 UniProtKB: Q9NYY1 4353 4354 4355, 4356, 4357, 4358,4359, 4360 IL-21 RefSeq: NP_001193935.1 4361 4362 4363, 4364, 4365,4366, 4367, 4368 IL-21 RefSeq: NP_068575.1 4369 4370 4371, 4372, 4373,4374, 4375, 4376 IL-22 UniProtKB: Q9GZX6 4377 4378 4379, 4380, 4381,4382, 4383, 4384 IL-23 UniProtKB: Q9NPF7 4385 4386 4387, 4388, 4389,4390, 4391, 4392 IL-24 UniProtKB: Q13007 4393 4394 4395, 4396, 4397,4398, 4399, 4400 IL-24 UniProtKB: Q2YHE6 4401 4402 4403, 4404, 4405,4406, 4407, 4408 IL-25 UniProtKB: Q969H8 4409 4410 4411, 4412, 4413,4414, 4415, 4416 IL-25 UniProtKB: Q9H293 4417 4418 4419, 4420, 4421,4422, 4423, 4424 IL-26 UniProtKB: Q9NPH9 4425 4426 4427, 4428, 4429,4430, 4431, 4432 IL-27A UniProtKB: Q8NEV9 4433 4434 4435, 4436, 4437,4438, 4439, 4440 IL-27B UniProtKB: Q14213 4441 4442 4443, 4444, 4445,4446, 4447, 4448 IL-28A UniProtKB: Q8IZJ0 4449 4450 4451, 4452, 4453,4454, 4455, 4456 IL-28B UniProtKB: Q8IZI9 4457 4458 4459, 4460, 4461,4462, 4463, 4464 IL-29 UniProtKB: Q8IU54 4465 4466 4467, 4468, 4469,4470, 4471, 4472 IL-2 UniProtKB: P60568 4473 4474 4475, 4476, 4477,4478, 4479, 4480 IL-2 UniProtKB: Q0GK43 4481 4482 4483, 4484, 4485,4486, 4487, 4488 IL-2 UniProtKB: Q13169 4489 4490 4491, 4492, 4493,4494, 4495, 4496 IL-2 UniProtKB: Q6NZ91 4497 4498 4499, 4500, 4501,4502, 4503, 4504 IL-2 UniProtKB: Q6NZ93 4505 4506 4507, 4508, 4509,4510, 4511, 4512 IL-31 UniProtKB: Q6EBC2 4513 4514 4515, 4516, 4517,4518, 4519, 4520 IL-32 UniProtKB: P24001 4521 4522 4523, 4524, 4525,4526, 4527, 4528 IL-33 UniProtKB: O95760 4529 4530 4531, 4532, 4533,4534, 4535, 4536 IL-37 UniProtKB: Q9NZH6 4537 4538 4539, 4540, 4541,4542, 4543, 4544 IL-3 UniProtKB: P08700 4545 4546 4547, 4548, 4549,4550, 4551, 4552 IL-3 UniProtKB: Q6NZ78 4553 4554 4555, 4556, 4557,4558, 4559, 4560 IL-3 UniProtKB: Q6NZ79 4561 4562 4563, 4564, 4565,4566, 4567, 4568 IL-4 UniProtKB: P05112-2 4569 4570 4571, 4572, 4573,4574, 4575, 4576 IL-4 UniProtKB: P05112 4577 4578 4579, 4580, 4581,4582, 4583, 4584 IL-5 UniProtKB: P05113 4585 4586 4587, 4588, 4589,4590, 4591, 4592 IL-6 UniProtKB: P05231 4593 4594 4595, 4596, 4597,4598, 4599, 4600 IL-7 UniProtKB: A8K673 4601 4602 4603, 4604, 4605,4606, 4607, 4608 IL-7 UniProtKB: P13232 4609 4610 4611, 4612, 4613,4614, 4615, 4616 IL-9 UniProtKB: P15248 4617 4618 4619, 4620, 4621,4622, 4623, 4624 LIF UniProtKB: P15018 4625 4626 4627, 4628, 4629, 4630,4631, 4632 LIGHT UniProtKB: O43557 4633 4634 4635, 4636, 4637, 4638,4639, 4640 LTalpha UniProtKB: B4DVZ8 4641 4642 4643, 4644, 4645, 4646,4647, 4648 LTalpha UniProtKB: P01374 4649 4650 4651, 4652, 4653, 4654,4655, 4656 LTalpha UniProtKB: P09960 4657 4658 4659, 4660, 4661, 4662,4663, 4664 LTalpha UniProtKB: Q5ST95 4665 4666 4667, 4668, 4669, 4670,4671, 4672 LTalpha UniProtKB: Q5STV3 4673 4674 4675, 4676, 4677, 4678,4679, 4680 LTalpha UniProtKB: Q6FG55 4681 4682 4683, 4684, 4685, 4686,4687, 4688 LTbeta UniProtKB: Q06643 4689 4690 4691, 4692, 4693, 4694,4695, 4696 LTbeta UniProtKB: Q5STB2 4697 4698 4699, 4700, 4701, 4702,4703, 4704 M-CSF UniProtKB: P09603 4705 4706 4707, 4708, 4709, 4710,4711, 4712 MIF UniProtKB: A6MUU8 4713 4714 4715, 4716, 4717, 4718, 4719,4720 MIF UniProtKB: P14174 4721 4722 4723, 4724, 4725, 4726, 4727, 4728OSM UniProtKB: P13725 4729 4730 4731, 4732, 4733, 4734, 4735, 4736 OX40LUniProtKB: P23510 4737 4738 4739, 4740, 4741, 4742, 4743, 4744 OX40LUniProtKB: P43489 4745 4746 4747, 4748, 4749, 4750, 4751, 4752RANK_ligand UniProtKB: O14788 4753 4754 4755, 4756, 4757, 4758, 4759,4760 Stem_Cell_Factor UniProtKB: P21583-2 4761 4762 4763, 4764, 4765,4766, 4767, 4768 Stem_Cell_Factor UniProtKB: P21583 4769 4770 4771,4772, 4773, 4774, 4775, 4776 TGFbeta1 UniProtKB: A0A024R0P8 4777 47784779, 4780, 4781, 4782, 4783, 4784 TGFbeta1 UniProtKB: P01137 4785 47864787, 4788, 4789, 4790, 4791, 4792 TGFbeta2 UniProtKB: P61812 4793 47944795, 4796, 4797, 4798, 4799, 4800 TGFbeta3 UniProtKB: A5YM40 4801 48024803, 4804, 4805, 4806, 4807, 4808 TGFbeta3 UniProtKB: P10600 4809 48104811, 4812, 4813, 4814, 4815, 4816 TL1A UniProtKB: O95150 4817 48184819, 4820, 4821, 4822, 4823, 4824 TWEAK UniProtKB: Q4ACW9 4825 48264827, 4828, 4829, 4830, 4831, 4832 CXCL8 UniProtKB: P10145 5265 52665267, 5268, 5269, 5270, 5271, 5272 Apo2L/TRAIL UniProtKB: P50591 68976898 6899, 6900, 6901, 6902, 6903, 6904 FasL UniProtKB: P48023 7321 73227323, 7324, 7325, 7326, 7327, 7328 TNF UniProtKB: P01375 7369 7370 7371,7372, 7373, 7374, 7375, 7376 TNF UniProtKB: Q5STB3 7377 7378 7379, 7380,7381, 7382, 7383, 7384

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one cytokine ora fragment or variant thereof, wherein the at least one coding regioncomprises an RNA sequence being identical or at least 50%, 60%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequencesaccording to the SEQ ID Nos as disclosed in Table 1.

2. Chemokines:

In a further preferred embodiment of the inventive RNA containingcomposition the RNA comprises at least one coding region that codes forat least one chemokine, or a fragment or variant thereof.

Chemokines are chemotactic cytokines that control the migratory patternsand positioning of immune cells, as reviewed by Griffith et al., 2014.Annu. Rev. Immunol. 32:659-702 (PMID 24655300). Chemokine function iscritical for all immune cell movement ranging from the migrationrequired for immune cell development and homeostasis, to that requiredfor the generation of primary and amnestic cellular and humoral immuneresponses, to the pathologic recruitment of immune cells in disease.Chemokines constitute the largest family of cytokines, consisting ofapproximately 50 endogenous chemokine ligands in humans and mice.

According to preferred embodiments in the context of the presentinvention chemokines may be selected from any chemokine selected fromthe group consisting of CCL1; CCL11; CCL12; CCL13; CCL14; CCL15; CCL16;CCL17; CCL18; CCL19; CCL2; CCL20; CCL21; CCL22; CCL24; CCL25; CCL26;CCL27; CCL28; CCL3; CCL4; CCL5; CCL6; CCL7; CCL8; CCL9; CX3CL1; CXCL1;CXCL10; CXCL11; CXCL12; CXCL13; CXCL14; CXCL15; CXCL2; CXCL3; CXCL4;CXCL5; CXCL6; CXCL7; CXCL8; CXCL9; XCL1; XCL2, preferably as disclosedin Table 2. Particularly preferred in this context are the RNA sequencesencoding a chemokine according to Table 2.

TABLE 2 Chemokines RNA Protein Sequence Sequence wild type SEQ ID SEQ IDRNA Sequence Gene Name Protein Accession No. NO: NO: SEQ ID NO: CCL11UniProtKB: P51671 4833 4834 4835, 4836, 4837, 4838, 4839, 4840 CCL11UniProtKB: Q6I9T4 4841 4842 4843, 4844, 4845, 4846, 4847, 4848 CCL12UniProtKB: Q62401 4849 4850 4851, 4852, 4853, 4854, 4855, 4856 CCL13UniProtKB: Q99616 4857 4858 4859, 4860, 4861, 4862, 4863, 4864 CCL14UniProtKB: Q16627 4865 4866 4867, 4868, 4869, 4870, 4871, 4872 CCL15UniProtKB: Q16663 4873 4874 4875, 4876, 4877, 4878, 4879, 4880 CCL16UniProtKB: O15467 4881 4882 4883, 4884, 4885, 4886, 4887, 4888 CCL17UniProtKB: Q92583 4889 4890 4891, 4892, 4893, 4894, 4895, 4896 CCL18UniProtKB: P55774 4897 4898 4899, 4900, 4901, 4902, 4903, 4904 CCL19UniProtKB: Q6IBD6 4905 4906 4907, 4908, 4909, 4910, 4911, 4912 CCL19UniProtKB: Q99731 4913 4914 4915, 4916, 4917, 4918, 4919, 4920 CCL1UniProtKB: P22362 4921 4922 4923, 4924, 4925, 4926, 4927, 4928 CCL20UniProtKB: P78556 4929 4930 4931, 4932, 4933, 4934, 4935, 4936 CCL21UniProtKB: O00585 4937 4938 4939, 4940, 4941, 4942, 4943, 4944 CCL22UniProtKB: O00626 4945 4946 4947, 4948, 4949, 4950, 4951, 4952 CCL24UniProtKB: O00175 4953 4954 4955, 4956, 4957, 4958, 4959, 4960 CCL25UniProtKB: O15444 4961 4962 4963, 4964, 4965, 4966, 4967, 4968 CCL26UniProtKB: Q9Y258 4969 4970 4971, 4972, 4973, 4974, 4975, 4976 CCL27UniProtKB: Q5VZ77 4977 4978 4979, 4980, 4981, 4982, 4983, 4984 CCL28UniProtKB: A0N0Q3 4985 4986 4987, 4988, 4989, 4990, 4991, 4992 CCL28UniProtKB: Q9NRJ3 4993 4994 4995, 4996, 4997, 4998, 4999, 5000 CCL2UniProtKB: P13500 5001 5002 5003, 5004, 5005, 5006, 5007, 5008 CCL3UniProtKB: A0N0R1 5009 5010 5011, 5012, 5013, 5014, 5015, 5016 CCL3UniProtKB: P10147 5017 5018 5019, 5020, 5021, 5022, 5023, 5024 CCL4UniProtKB: P13236 5025 5026 5027, 5028, 5029, 5030, 5031, 5032 CCL4UniProtKB: Q7M4M2 5033 5034 5035, 5036, 5037, 5038, 5039, 5040 CCL5UniProtKB: D0EI67 5041 5042 5043, 5044, 5045, 5046, 5047, 5048 CCL5UniProtKB: P13501 5049 5050 5051, 5052, 5053, 5054, 5055, 5056 CCL6UniProtKB: P27784 5057 5058 5059, 5060, 5061, 5062, 5063, 5064 CCL7UniProtKB: P80098 5065 5066 5067, 5068, 5069, 5070, 5071, 5072 CCL7UniProtKB: Q7Z7Q8 5073 5074 5075, 5076, 5077, 5078, 5079, 5080 CCL8UniProtKB: H0UIC7 5081 5082 5083, 5084, 5085, 5086, 5087, 5088 CCL8UniProtKB: P80075 5089 5090 5091, 5092, 5093, 5094, 5095, 5096 CCL9UniProtKB: P51670 5097 5098 5099, 5100, 5101, 5102, 5103, 5104 CX3CL1UniProtKB: A0N0N7 5105 5106 5107, 5108, 5109, 5110, 5111, 5112 CX3CL1UniProtKB: P78423 5113 5114 5115, 5116, 5117, 5118, 5119, 5120 CX3CL1UniProtKB: Q6I9S9 5121 5122 5123, 5124, 5125, 5126, 5127, 5128 CXCL10UniProtKB: A0A024RDA4 5129 5130 5131, 5132, 5133, 5134, 5135, 5136CXCL10 UniProtKB: P02778 5137 5138 5139, 5140, 5141, 5142, 5143, 5144CXCL11 UniProtKB: O14625 5145 5146 5147, 5148, 5149, 5150, 5151, 5152CXCL12 UniProtKB: P48061 5153 5154 5155, 5156, 5157, 5158, 5159, 5160CXCL13 UniProtKB: L8E878 5161 5162 5163, 5164, 5165, 5166, 5167, 5168CXCL13 UniProtKB: O43927 5169 5170 5171, 5172, 5173, 5174, 5175, 5176CXCL14 UniProtKB: O95715 5177 5178 5179, 5180, 5181, 5182, 5183, 5184CXCL15 UniProtKB: Q9WVL7 5185 5186 5187, 5188, 5189, 5190, 5191, 5192CXCL1 UniProtKB: P09341 5193 5194 5195, 5196, 5197, 5198, 5199, 5200CXCL2 UniProtKB: A0A024RDD9 5201 5202 5203, 5204, 5205, 5206, 5207, 5208CXCL2 UniProtKB: P19875 5209 5210 5211, 5212, 5213, 5214, 5215, 5216CXCL3 UniProtKB: P19876 5217 5218 5219, 5220, 5221, 5222, 5223, 5224CXCL4 UniProtKB: P02776 5225 5226 5227, 5228, 5229, 5230, 5231, 5232CXCL5 UniProtKB: P42830 5233 5234 5235, 5236, 5237, 5238, 5239, 5240CXCL5 UniProtKB: Q6I9S7 5241 5242 5243, 5244, 5245, 5246, 5247, 5248CXCL6 UniProtKB: P80162 5249 5250 5251, 5252, 5253, 5254, 5255, 5256CXCL7 UniProtKB: P02775 5257 5258 5259, 5260, 5261, 5262, 5263, 5264CXCL8 UniProtKB: P10145 5265 5266 5267, 5268, 5269, 5270, 5271, 5272CXCL9 UniProtKB: L8E8X0 5273 5274 5275, 5276, 5277, 5278, 5279, 5280CXCL9 UniProtKB: Q07325 5281 5282 5283, 5284, 5285, 5286, 5287, 5288XCL1 UniProtKB: P47992 5289 5290 5291, 5292, 5293, 5294, 5295, 5296 XCL2UniProtKB: Q9UBD3 5297 5298 5299, 5300, 5301, 5302, 5303, 5304

In this context particularly preferred are chemokines chosen from thefollowing list: CXCL9, CXCL10, CCL5, XCL1, CCL20, CCL19, CCL21, CCL2,CCL3, CCL16, and CXCL12.

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one chemokine ora fragment or variant thereof, wherein the at least one coding regioncomprises an RNA sequence being identical or at least 50%, 60%, 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNA sequencesaccording to the SEQ ID Nos as disclosed in Table 2.

3. Suicide Gene Products

In a further preferred embodiment of the inventive RNA containingcomposition the RNA codes for at least one so-called suicide geneproduct, especially for a suicide enzyme, preferably for a nucleotidemetabolizing enzyme. Preferably the RNA is used in combination with aprodrug which is a substrate of the suicide gene product, especially thesuicide enzyme, and which is converted to a cytotoxic compound by thesuicide gene product. The appropriate prodrug may be added to theinventive RNA composition or it may be administered separately to thepatient.

One or more preferred suicide enzymes may be chosen from the followinglist: thymidine kinase, preferably a viral thymidine kinase, morepreferrably Herpes simplex virus thymidine kinase, Varicella zosterthymidine kinase; a plant thymidine kinase, preferably a tomatothymidine kinase; cytosine deaminase, preferably bacterial cytosinedeaminase or Yeast cytosine deaminase; deoxynucleoside kinase,preferably Drosophila melanogaster deoxynucleoside kinase; deoxycytidinekinase, preferably a mammalian deoxycytidine kinase, purine nucleosidephosphorylase, preferably a bacterial purine nucleoside phosphorylase.

It is already known that suicide gene therapy is a promising treatmentfor cancer (Ardiani et al., 2012. Curr. Gene Ther. 12(2):77-91. PMID:22384805). This approach is based on the successful delivery andexpression of the suicide gene in tumor cells. The suicide gene encodesan enzyme with the unique ability to activate an otherwise ineffectiveprodrug. Following suicide gene expression in transfected cells, anappropriate prodrug is administered and is converted to a cytotoxiccompound by the actions of the suicide gene product. As most suicidegenes encode enzymes belonging to the class of nucleotide metabolizingenzymes, the general mode of action of activated prodrugs isinterference with DNA synthesis that consequently results in inductionof apoptosis. The potency of these drugs is maximized in cancer cellsdue to their greater proliferative rate as compared to normal cells.Because of the prospect to preferentially deliver genes to tumor cells,this strategy has the potential to offer selective tumor killing whilesparing normal cells, a feature that standard chemotherapeutic andradiotherapy approaches do not generally afford.

The following table 3 (Ardiani et al., 2012. Curr. Gene Ther.12(2):77-91. PMID: 22384805) summarizes preferred nucleotidemetabolizing enzymes usable in the inventive approach. The tableincludes variants and mutants of such enzymes which were generated byprotein engineering strategies.

TABLE 3 Suicide enzymes Drug Natural Variants/ inhibitors Enzyme Sourcegene substrate Prodrug Mutants action* Herpes Herpes ThymidineGanciclovir (GCV), Mutant 30 1 Simplex Simplex Virus 1 acyclovir (ACV)Mutant 75 1 Virus (HSV-1) SR39 1 Thymidine Thymidine A168H 1 KinaseKinase (TK) A167Y 1 (HSVTK) Q125N 1, 2 Bacterial Escherichia coli -Cytosine 5-Fluorocytosine D314 1, 2, 4 Cytosine codA (5-FC) mutantsDeaminase bCD1525 1, 2, 4 (bCD) Yeast Saccharomyces Cytosine 5-FC yCDtriple 1, 2, 4 Cytosine cerevisiae - D92E 1, 2, 4 Deaminase fcy1 (yCD)Drosophila Drosophila melanogaster - All four azidothymidine MuD 1, 5melanogaster dNK deoxy-ribo- (AZT), B5 1 Deoxynucleoside nucleo-dideoxycytoinse B10 1, 3 Kinase sides (ddC); M88R 1 (Dm-dNK) CdA;HDHD-12, 1, 5 9-beta-D- HD-16 arabinofuranosyl- R4.V3 1 2-fluoroadenine(F-AraA); GCV, 9-beta-D- arabinosylguanine (AraG); 2′,3′- didehydro-3′-deoxythymidine (D4T); 2′,3′- Dideoxythymidine (ddT) Deoxy- Homosapiens - Deoxy- 2′,2′-difluoro- DMMA, 1, 3 cytidine dCK cytidinedeoxycytidine DMLA Kinase (dCK) (dFdC), AraA, β-L- EpTK6 1, 3, 5thymidine (L-dT) Ser-74 1, 3 AZT cytarabine 5′- monophosphate (AraC)Purine Escherichia coli - Purine ribo- 9-(6-deoxy-α-L- M64V 1, 4Nucleoside deoD nucleo- talofuranosyl)-6- Phosphor- sides methylpurineylase (PNP) (Me(talo)-MeP-R) *Drug inhibitory action. 1: DNA synthesis;2: Thymidylate synthetase; 3: Ribonucleotide reductase; 4: RNA/proteinsynthesis; 5: Reverse transcriptase.

Herpes simplex virus type 1 thymidine kinase (HSVTK, EC 2.7.1.21), ahomodimer with a subunit molecular mass of 45 kDa, is responsible forthe phosphorylation of thymidine, deoxycytidine, deoxythymidylate (dTMP)as well as various pharmaceutically important pyrimidine and guanosineanalogs. Of particular note, HSVTK is responsible for the initial andrate limiting phosphorylation of the antiviral guanosine analogsacyclovir (ACV) and ganciclovir (GCV). Once monophosphorylated theseanalogs can be further phosphorylated by endogenous enzymes (guanylatekinase and nucleoside diphosphokinase) before being incorporated intonascent DNA to cause double strand destabilization and, subsequently,cell death.

Moreover, the Varicella zoster virus thymidine kinase (VZV-tk) may beused e.g. in conjunction with the prodrug 6-methoxypurine arabinoside(ara-M) or 1-(2′-deoxy-2-flioro-b-D-arabinofuranosyl)-5-iodouracil(FIAU). Other examples are thymidine kinases of Aleutian disease virus(ADV), respiratory syncytial virus (RSV) and cytomegalovirus (CMV).

Cytosine deaminase (CD; EC 3.5.4.1) is an enzyme in the pyrimidinesalvage pathway that catalyzes the deamination of cytosine to formuracil and ammonia. CD from E. coli (bCD) is a hexamer of 48 kDAsubunits with a catalytic metal iron. This enzyme is absent in mammalsand uniquely present in fungi and bacteria. It is used in suicide genetherapy because of its ability to deaminate the anti-fungal drug,5-fluorocytosine (5FC), to 5-fluorouracil (5FU), a potentanti-neoplastic drug. UPRT (Uracil phosphoribosyltransferase) may beused as potential enhancer.

Saccharomyces cerevisiae or Yeast cytosine deaminase (yCD, EC 3.5.4.1)is a homodimer of 17.5 kDa subunits and has been shown to be more activetowards 5FC than bCD (22-fold lower K_(m)) with a slightly bettercatalytic efficiency (k_(cat)/K_(m)) toward 5FC relative to its naturalsubstrate cytosine.

Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK; EC2.7.1.145) is a 29 kDa homodimeric, multisubstrate kinase able tophosphorylate all four natural deoxyribonucleosides required for DNAsynthesis. In addition to its broad substrate specificity, Dm-dNKexhibits higher catalytic rates toward these natural deoxynucleosidesand several nucleoside analogs as compared to mammalian deoxynucleosidekinases. Due to these distinctive characteristics Dm-dNK is a especiallypreferred enzyme for the inventive suicide gene therapy application.

Human deoxycytidine kinase (dCK; EC 2.7.1.74) is a 30.5 kDa homodimericenzyme in the salvage pathway of deoxyribonucleosides and is responsiblefor activating all natural deoxyribonucleosides, excluding thymidine, asprecursors for DNA synthesis. Due to its broad substrate specificity,dCK is able to activate multiple nucleoside analogs effective againstdifferent types of cancer. However, wild type dCK is intrinsically arelatively poor catalyst with low turnover rates and prodrug activationis dependent on its expression levels. Indeed, nucleoside analogs thatare efficient substrates of dCK, such as cytarabine (AraC), fludarabine(F-AraA), cladribine (CdA), and gemcitabine (dFdC), are effectiveanti-leukemic agents as lymphoblasts have been shown to have high dCKexpression levels whereas cancer cells lacking dCK activity areresistant to these same analogs. Therefore dCK is an especiallypreferred enzyme for the inventive approach.

Preferably the RNA of the inventive RNA containing composition is usedin combination with further components which enhance the cytotoxiceffect of the treatment. It is especially preferred to use the RNA incombination with RNA coding for connexins and/or with a protein of theconnexin family or parts or fragments thereof. Connexins aretransmembrane proteins which form gap junctions between cells. Theyallow transfer of e.g. molecules between neighboring cells therebyenabling the transfer of cytoxic substances.

Although suicide gene therapy is a fairly new anti-cancer approach, theconcept was originally described more than two decades ago in 1986 byMoolten (Moolten, 1986. Cancer Res. 46(10):5276-81). He also proposedthe existence of what is currently known as the bystander effect, nowwidely recognized as a fundamental feature of suicide gene therapysuccess. By definition the bystander effect is the extension ofcytotoxic effects from transfected cells to non-transfected neighboringcells such that complete tumor regression is observed when only a smallsubpopulation of tumor cells is successfully transfected. Thisphenomenon is crucial to the overall effectiveness of suicide genetherapy today due to low transfection efficiencies achievable byavailable delivery systems.

The bystander effect is thought to occur via two major mechanisms: localand immune-mediated. The local mechanism involves the killing ofuntransfected nearby cells due to the transfer of toxic materials orsuicide enzymes through gap junctions, apoptotic vesicles or throughdiffusion of soluble toxic metabolites. Gap junctions are important incell-cell interactions and are responsible for the transfer of ions,nucleotides and small molecules to adjacent cells. The transfer of toxicdrugs through gap junctions, however, may not be available in certaintypes of tumors that down regulate intracellular gap junctioncommunication and display disorganized and non-functional gap junctions.To address this problem, several studies have increased the expressionof connexins, the building blocks of gap junctions, and demonstratedthat enhanced bystander and cell killing effects were achieved.

Accumulating evidence from in vivo experiments suggests that theimmune-mediated bystander effect plays an important role in tumorregression as well. The presence of inflammatory infiltrates,chemokines, and cytokines have been found elevated in regressing tumorsof immune competent animals receiving suicide gene therapy treatment.These cytokines and chemokines further induce the production ofimmune-regulatory molecules able to stimulate a more robust anti-cancereffect and additionally, because death of transfected cells is throughapoptosis, numerous inflammatory signals may be released to evoke apotent immune response. Therefore the combination of the inventivecomposition with connexins or with RNA coding for connexins isespecially preferred, because it strengthens the bystander effectthereby increasing the efficiency of the inventive RNA containingcomposition.

According to preferred embodiments in the context of the presentinvention suicide gene products may be selected from any suicide geneproduct selected from the group consisting of Cytosine_Deaminase_codA;Cytosine_Deaminase_fcy1; Deoxy-cytidine_Kinase_dCK;Deoxynucleoside_Kinase_dNK; Purine_Nucleoside_Phosphorylase_deoD;Thymidine_Kinase_TK, preferably as disclosed in Table 4. Particularlypreferred in this context are the RNA sequences encoding a suicide geneproduct according to Table 4.

TABLE 4 Suicide Gene Products RNA Protein Sequence RNA Protein Sequencewild type Sequence Gene Name Accession No. SEQ ID NO: SEQ ID NO: SEQ IDNO: Cytosine_Deaminase_codA UniProtKB: 5305 5306 5307, 5308, 5309,A0A024KS17 5310, 5311, 5312 Cytosine_Deaminase_codA UniProtKB: 5313 53145315, 5316, 5317, A0A0H2V4N7 5318, 5319, 5320 Cytosine_Deaminase_codAUniProtKB: 5321 5322 5323, 5324, 5325, A0A0H2YX33 5326, 5327, 5328Cytosine_Deaminase_codA UniProtKB: 5329 5330 5331, 5332, 5333, F4NM905334, 5335, 5336 Cytosine_Deaminase_codA UniProtKB: 5337 5338 5339,5340, 5341, H9UNZ4 5342, 5343, 5344 Cytosine_Deaminase_codA UniProtKB:5345 5346 5347, 5348, 5349, Q1RFJ5 5350, 5351, 5352Cytosine_Deaminase_codA UniProtKB: 5353 5354 5355, 5356, 5357, Q2VP095358, 5359, 5360 Cytosine_Deaminase_codA UniProtKB: 5361 5362 5363,5364, 5365, Q53ZC8 5366, 5367, 5368 Cytosine_Deaminase_codA UniProtKB:5369 5370 5371, 5372, 5373, Q6Q8Q1 5374, 5375, 5376Cytosine_Deaminase_codA UniProtKB: 5377 5378 5379, 5380, 5381, W8ZNH55382, 5383, 5384 Cytosine_Deaminase_fcy1 UniProtKB: 5385 5386 5387,5388, 5389, A0A023ZJG6 5390, 5391, 5392 Cytosine_Deaminase_fcy1UniProtKB: 5393 5394 5395, 5396, 5397, A0A024XGF7 5398, 5399, 5400Cytosine_Deaminase_fcy1 UniProtKB: 5401 5402 5403, 5404, 5405,A0A024XUW9 5406, 5407, 5408 Cytosine_Deaminase_fcy1 UniProtKB: 5409 54105411, 5412, 5413, A0A0C5ITD0 5414, 5415, 5416 Cytosine_Deaminase_fcy1UniProtKB: 5417 5418 5419, 5420, 5421, A0A0D4WVI5 5422, 5423, 5424Cytosine_Deaminase_fcy1 UniProtKB: 5425 5426 5427, 5428, 5429,A0A0D4WY08 5430, 5431, 5432 Cytosine_Deaminase_fcy1 UniProtKB: 5433 54345435, 5436, 5437, A0A0D4WZA2 5438, 5439, 5440 Cytosine_Deaminase_fcy1UniProtKB: 5441 5442 5443, 5444, 5445, A0A0D4WZQ5 5446, 5447, 5448Cytosine_Deaminase_fcy1 UniProtKB: 5449 5450 5451, 5452, 5453,A0A0D4X0R8 5454, 5455, 5456 Cytosine_Deaminase_fcy1 UniProtKB: 5457 54585459, 5460, 5461, A0A0D4X195 5462, 5463, 5464 Cytosine_Deaminase_fcy1UniProtKB: 5465 5466 5467, 5468, 5469, A0A0D4X2R9 5470, 5471, 5472Cytosine_Deaminase_fcy1 UniProtKB: 5473 5474 5475, 5476, 5477,A0A0D4X3Q1 5478, 5479, 5480 Cytosine_Deaminase_fcy1 UniProtKB: 5481 54825483, 5484, 5485, A0A0D4X4K1 5486, 5487, 5488 Cytosine_Deaminase_fcy1UniProtKB: 5489 5490 5491, 5492, 5493, A0A0D4X5B7 5494, 5495, 5496Cytosine_Deaminase_fcy1 UniProtKB: 5497 5498 5499, 5500, 5501,A0A0D4X7R4 5502, 5503, 5504 Cytosine_Deaminase_fcy1 UniProtKB: 5505 55065507, 5508, 5509, A0A0D4X7X4 5510, 5511, 5512 Cytosine_Deaminase_fcy1UniProtKB: 5513 5514 5515, 5516, 5517, A0A0D4XA07 5518, 5519, 5520Cytosine_Deaminase_fcy1 UniProtKB: 5521 5522 5523, 5524, 5525,A0A0D4XA25 5526, 5527, 5528 Cytosine_Deaminase_fcy1 UniProtKB: 5529 55305531, 5532, 5533, A0A0D4XAV6 5534, 5535, 5536 Cytosine_Deaminase_fcy1UniProtKB: 5537 5538 5539, 5540, 5541, A0A0D4XCJ5 5542, 5543, 5544Cytosine_Deaminase_fcy1 UniProtKB: 5545 5546 5547, 5548, 5549,A0A0D4XDL4 5550, 5551, 5552 Cytosine_Deaminase_fcy1 UniProtKB: 5553 55545555, 5556, 5557, A0A0D4XG53 5558, 5559, 5560 Cytosine_Deaminase_fcy1UniProtKB: 5561 5562 5563, 5564, 5565, A0A0D4XGH3 5566, 5567, 5568Cytosine_Deaminase_fcy1 UniProtKB: 5569 5570 5571, 5572, 5573,A0A0D4XHD4 5574, 5575, 5576 Cytosine_Deaminase_fcy1 UniProtKB: 5577 55785579, 5580, 5581, A0A0D4XIK5 5582, 5583, 5584 Cytosine_Deaminase_fcy1UniProtKB: 5585 5586 5587, 5588, 5589, A0A0D4XJR4 5590, 5591, 5592Cytosine_Deaminase_fcy1 UniProtKB: 5593 5594 5595, 5596, 5597,A0A0D4XL36 5598, 5599, 5600 Cytosine_Deaminase_fcy1 UniProtKB: 5601 56025603, 5604, 5605, A0A0D4XNH2 5606, 5607, 5608 Cytosine_Deaminase_fcy1UniProtKB: 5609 5610 5611, 5612, 5613, A0A0D4XNS1 5614, 5615, 5616Cytosine_Deaminase_fcy1 UniProtKB: 5617 5618 5619, 5620, 5621,A0A0D4XQY5 5622, 5623, 5624 Cytosine_Deaminase_fcy1 UniProtKB: 5625 56265627, 5628, 5629, A0A0D4XS80 5630, 5631, 5632 Cytosine_Deaminase_fcy1UniProtKB: 5633 5634 5635, 5636, 5637, A0A0D4XS82 5638, 5639, 5640Cytosine_Deaminase_fcy1 UniProtKB: 5641 5642 5643, 5644, 5645,A0A0D4XTC2 5646, 5647, 5648 Cytosine_Deaminase_fcy1 UniProtKB: 5649 56505651, 5652, 5653, A0A0D4XUZ4 5654, 5655, 5656 Cytosine_Deaminase_fcy1UniProtKB: 5657 5658 5659, 5660, 5661, A0A0D4XW26 5662, 5663, 5664Cytosine_Deaminase_fcy1 UniProtKB: 5665 5666 5667, 5668, 5669,A0A0D4XXD1 5670, 5671, 5672 Cytosine_Deaminase_fcy1 UniProtKB: 5673 56745675, 5676, 5677, A0A0D4XYH3 5678, 5679, 5680 Cytosine_Deaminase_fcy1UniProtKB: 5681 5682 5683, 5684, 5685, A0A0D4XZT0 5686, 5687, 5688Cytosine_Deaminase_fcy1 UniProtKB: 5689 5690 5691, 5692, 5693,A0A0D4Y164 5694, 5695, 5696 Cytosine_Deaminase_fcy1 UniProtKB: 5697 56985699, 5700, 5701, A0A0D4Y2A8 5702, 5703, 5704 Cytosine_Deaminase_fcy1UniProtKB: 5705 5706 5707, 5708, 5709, A0A0D4Y3N1 5710, 5711, 5712Cytosine_Deaminase_fcy1 UniProtKB: 5713 5714 5715, 5716, 5717,A0A0D4Y5S3 5718, 5719, 5720 Cytosine_Deaminase_fcy1 UniProtKB: 5721 57225723, 5724, 5725, A0A0D4Y5Y1 5726, 5727, 5728 Cytosine_Deaminase_fcy1UniProtKB: 5729 5730 5731, 5732, 5733, A0A0D4Y7I2 5734, 5735, 5736Cytosine_Deaminase_fcy1 UniProtKB: 5737 5738 5739, 5740, 5741,A0A0D4Y8S5 5742, 5743, 5744 Cytosine_Deaminase_fcy1 UniProtKB: 5745 57465747, 5748, 5749, A0A0D4YAR2 5750, 5751, 5752 Cytosine_Deaminase_fcy1UniProtKB: 5753 5754 5755, 5756, 5757, A0A0D4YBY2 5758, 5759, 5760Cytosine_Deaminase_fcy1 UniProtKB: 5761 5762 5763, 5764, 5765,A0A0D4YCB3 5766, 5767, 5768 Cytosine_Deaminase_fcy1 UniProtKB: 5769 57705771, 5772, 5773, A0A0D4YEC2 5774, 5775, 5776 Cytosine_Deaminase_fcy1UniProtKB: 5777 5778 5779, 5780, 5781, A0A0D4YF30 5782, 5783, 5784Cytosine_Deaminase_fcy1 UniProtKB: 5785 5786 5787, 5788, 5789,A0A0D4YGU2 5790, 5791, 5792 Cytosine_Deaminase_fcy1 UniProtKB: 5793 57945795, 5796, 5797, A0A0D4YHN3 5798, 5799, 5800 Cytosine_Deaminase_fcy1UniProtKB: 5801 5802 5803, 5804, 5805, A0A0D4YIU4 5806, 5807, 5808Cytosine_Deaminase_fcy1 UniProtKB: 5809 5810 5811, 5812, 5813,A0A0D4YJ74 5814, 5815, 5816 Cytosine_Deaminase_fcy1 UniProtKB: 5817 58185819, 5820, 5821, A0A0D4YKC5 5822, 5823, 5824 Cytosine_Deaminase_fcy1UniProtKB: 5825 5826 5827, 5828, 5829, A0A0D4YMN8 5830, 5831, 5832Cytosine_Deaminase_fcy1 UniProtKB: 5833 5834 5835, 5836, 5837,A0A0D4YMV6 5838, 5839, 5840 Cytosine_Deaminase_fcy1 UniProtKB: 5841 58425843, 5844, 5845, A0A0D4YPP6 5846, 5847, 5848 Cytosine_Deaminase_fcy1UniProtKB: 5849 5850 5851, 5852, 5853, A0A0D4YRD4 5854, 5855, 5856Cytosine_Deaminase_fcy1 UniProtKB: 5857 5858 5859, 5860, 5861,A0A0D4YS13 5862, 5863, 5864 Cytosine_Deaminase_fcy1 UniProtKB: 5865 58665867, 5868, 5869, A0A0D4YTJ7 5870, 5871, 5872 Cytosine_Deaminase_fcy1UniProtKB: 5873 5874 5875, 5876, 5877, A0A0D4YUX9 5878, 5879, 5880Cytosine_Deaminase_fcy1 UniProtKB: 5881 5882 5883, 5884, 5885,A0A0D4YV34 5886, 5887, 5888 Cytosine_Deaminase_fcy1 UniProtKB: 5889 58905891, 5892, 5893, A0A0D4YXE1 5894, 5895, 5896 Cytosine_Deaminase_fcy1UniProtKB: 5897 5898 5899, 5900, 5901, A0A0D4YYM6 5902, 5903, 5904Cytosine_Deaminase_fcy1 UniProtKB: 5905 5906 5907, 5908, 5909,A0A0D4YZB7 5910, 5911, 5912 Cytosine_Deaminase_fcy1 UniProtKB: 5913 59145915, 5916, 5917, A0A0D4Z060 5918, 5919, 5920 Cytosine_Deaminase_fcy1UniProtKB: 5921 5922 5923, 5924, 5925, A0A0D4Z1S2 5926, 5927, 5928Cytosine_Deaminase_fcy1 UniProtKB: 5929 5930 5931, 5932, 5933,A0A0D4Z2L6 5934, 5935, 5936 Cytosine_Deaminase_fcy1 UniProtKB: 5937 59385939, 5940, 5941, A0A0D4Z4A1 5942, 5943, 5944 Cytosine_Deaminase_fcy1UniProtKB: 5945 5946 5947, 5948, 5949, A0A0D4Z552 5950, 5951, 5952Cytosine_Deaminase_fcy1 UniProtKB: 5953 5954 5955, 5956, 5957,A0A0D4Z6N6 5958, 5959, 5960 Cytosine_Deaminase_fcy1 UniProtKB: 5961 59625963, 5964, 5965, A0A0D4Z800 5966, 5967, 5968 Cytosine_Deaminase_fcy1UniProtKB: 5969 5970 5971, 5972, 5973, A0A0D4Z9V2 5974, 5975, 5976Cytosine_Deaminase_fcy1 UniProtKB: 5977 5978 5979, 5980, 5981,A0A0D4ZB52 5982, 5983, 5984 Cytosine_Deaminase_fcy1 UniProtKB: 5985 59865987, 5988, 5989, A0A0D4ZCA2 5990, 5991, 5992 Cytosine_Deaminase_fcy1UniProtKB: 5993 5994 5995, 5996, 5997, A0A0D4ZCG3 5998, 5999, 6000Cytosine_Deaminase_fcy1 UniProtKB: 6001 6002 6003, 6004, 6005,A0A0D4ZEM2 6006, 6007, 6008 Cytosine_Deaminase_fcy1 UniProtKB: 6009 60106011, 6012, 6013, A0A0D4ZFD0 6014, 6015, 6016 Cytosine_Deaminase_fcy1UniProtKB: 6017 6018 6019, 6020, 6021, A0A0D4ZGR1 6022, 6023, 6024Cytosine_Deaminase_fcy1 UniProtKB: 6025 6026 6027, 6028, 6029,A0A0D4ZIM2 6030, 6031, 6032 Cytosine_Deaminase_fcy1 UniProtKB: 6033 60346035, 6036, 6037, A0A0D4ZJC0 6038, 6039, 6040 Cytosine_Deaminase_fcy1UniProtKB: 6041 6042 6043, 6044, 6045, A0A0D4ZK17 6046, 6047, 6048Cytosine_Deaminase_fcy1 UniProtKB: 6049 6050 6051, 6052, 6053,A0A0D4ZMC8 6054, 6055, 6056 Cytosine_Deaminase_fcy1 UniProtKB: 6057 60586059, 6060, 6061, A0A0D4ZMX9 6062, 6063, 6064 Cytosine_Deaminase_fcy1UniProtKB: 6065 6066 6067, 6068, 6069, A0A0D4ZP21 6070, 6071, 6072Cytosine_Deaminase_fcy1 UniProtKB: 6073 6074 6075, 6076, 6077,A0A0D4ZQ62 6078, 6079, 6080 Cytosine_Deaminase_fcy1 UniProtKB: 6081 60826083, 6084, 6085, A0A0D4ZQ92 6086, 6087, 6088 Cytosine_Deaminase_fcy1UniProtKB: 6089 6090 6091, 6092, 6093, A0A0D4ZS31 6094, 6095, 6096Cytosine_Deaminase_fcy1 UniProtKB: 6097 6098 6099, 6100, 6101,A0A0D4ZS87 6102, 6103, 6104 Cytosine_Deaminase_fcy1 UniProtKB: 6105 61066107, 6108, 6109, A0A0D4ZTS6 6110, 6111, 6112 Cytosine_Deaminase_fcy1UniProtKB: 6113 6114 6115, 6116, 6117, A0A0D4ZUK0 6118, 6119, 6120Cytosine_Deaminase_fcy1 UniProtKB: 6121 6122 6123, 6124, 6125,A0A0D4ZVN6 6126, 6127, 6128 Cytosine_Deaminase_fcy1 UniProtKB: 6129 61306131, 6132, 6133, A0A0D4ZWP2 6134, 6135, 6136 Cytosine_Deaminase_fcy1UniProtKB: 6137 6138 6139, 6140, 6141, A0A0D4ZX07 6142, 6143, 6144Cytosine_Deaminase_fcy1 UniProtKB: 6145 6146 6147, 6148, 6149, Q121786150, 6151, 6152 Cytosine_Deaminase_fcy1 UniProtKB: 6153 6154 6155,6156, 6157, W7PK48 6158, 6159, 6160 Cytosine_Deaminase_fcy1 UniProtKB:6161 6162 6163, 6164, 6165, W7R647 6166, 6167, 6168Deoxy-cytidine_Kinase_dCK UniProtKB: 6169 6170 6171, 6172, 6173, P277076174, 6175, 6176 Deoxynucleoside_Kinase_dNK UniProtKB: 6177 6178 6179,6180, 6181, Q540Z9 6182, 6183, 6184 Deoxynucleoside_Kinase_dNKUniProtKB: 6185 6186 6187, 6188, 6189, Q9XZT6 6190, 6191, 6192Purine_Nucleoside_(—) UniProtKB: 6193 6194 6195, 6196, 6197,Phosphorylase_deoD A0A023Z7B9 6198, 6199, 6200 Purine_Nucleoside_(—)UniProtKB: 6201 6202 6203, 6204, 6205, Phosphorylase_deoD A0A024KMI26206, 6207, 6208 Purine_Nucleoside_(—) UniProtKB: 6209 6210 6211, 6212,6213, Phosphorylase_deoD A0A0E0SRY5 6214, 6215, 6216Purine_Nucleoside_(—) UniProtKB: 6217 6218 6219, 6220, 6221,Phosphorylase_deoD A0A0E0U7I4 6222, 6223, 6224 Purine_Nucleoside_(—)UniProtKB: 6225 6226 6227, 6228, 6229, Phosphorylase_deoD A0A0E0VFI36230, 6231, 6232 Purine_Nucleoside_(—) UniProtKB: 6233 6234 6235, 6236,6237, Phosphorylase_deoD A0A0E0Y455 6238, 6239, 6240Purine_Nucleoside_(—) UniProtKB: 6241 6242 6243, 6244, 6245,Phosphorylase_deoD A0A0E1M7E2 6246, 6247, 6248 Purine_Nucleoside_(—)UniProtKB: 6249 6250 6251, 6252, 6253, Phosphorylase_deoD A0A0E3KJD76254, 6255, 6256 Purine_Nucleoside_(—) UniProtKB: 6257 6258 6259, 6260,6261, Phosphorylase_deoD A0A0F6CCW6 6262, 6263, 6264Purine_Nucleoside_(—) UniProtKB: 6265 6266 6267, 6268, 6269,Phosphorylase_deoD A0A0F6FGI8 6270, 6271, 6272 Purine_Nucleoside_(—)UniProtKB: 6273 6274 6275, 6276, 6277, Phosphorylase_deoD A0A0F6GWR26278, 6279, 6280 Purine_Nucleoside_(—) UniProtKB: 6281 6282 6283, 6284,6285, Phosphorylase_deoD A0A0G2SIK5 6286, 6287, 6288Purine_Nucleoside_(—) UniProtKB: 6289 6290 6291, 6292, 6293,Phosphorylase_deoD A0A0G3J9R6 6294, 6295, 6296 Purine_Nucleoside_(—)UniProtKB: 6297 6298 6299, 6300, 6301, Phosphorylase_deoD A0A0G3J9Y26302, 6303, 6304 Purine_Nucleoside_(—) UniProtKB: 6305 6306 6307, 6308,6309, Phosphorylase_deoD A0A0G3KD68 6310, 6311, 6312Purine_Nucleoside_(—) UniProtKB: 6313 6314 6315, 6316, 6317,Phosphorylase_deoD A0A0H2Z6H1 6318, 6319, 6320 Purine_Nucleoside_(—)UniProtKB: 6321 6322 6323, 6324, 6325, Phosphorylase_deoD A0A0H3EQW16326, 6327, 6328 Purine_Nucleoside_(—) UniProtKB: 6329 6330 6331, 6332,6333, Phosphorylase_deoD A0A0H3XF09 6334, 6335, 6336Purine_Nucleoside_(—) UniProtKB: 6337 6338 6339, 6340, 6341,Phosphorylase_deoD A0A0J9WZC9 6342, 6343, 6344 Purine_Nucleoside_(—)UniProtKB: 6345 6346 6347, 6348, 6349, Phosphorylase_deoD A7ZVS7 6350,6351, 6352 Purine_Nucleoside_(—) UniProtKB: 6353 6354 6355, 6356, 6357,Phosphorylase_deoD A8A8B3 6358, 6359, 6360 Purine_Nucleoside_(—)UniProtKB: 6361 6362 6363, 6364, 6365, Phosphorylase_deoD B1IS35 6366,6367, 6368 Purine_Nucleoside_(—) UniProtKB: 6369 6370 6371, 6372, 6373,Phosphorylase_deoD B1LEI9 6374, 6375, 6376 Purine_Nucleoside_(—)UniProtKB: 6377 6378 6379, 6380, 6381, Phosphorylase_deoD B1XFJ4 6382,6383, 6384 Purine_Nucleoside_(—) UniProtKB: 6385 6386 6387, 6388, 6389,Phosphorylase_deoD B3HEI4 6390, 6391, 6392 Purine_Nucleoside_(—)UniProtKB: 6393 6394 6395, 6396, 6397, Phosphorylase_deoD B5Z4R6 6398,6399, 6400 Purine_Nucleoside_(—) UniProtKB: 6401 6402 6403, 6404, 6405,Phosphorylase_deoD B6I6N1 6406, 6407, 6408 Purine_Nucleoside_(—)UniProtKB: 6409 6410 6411, 6412, 6413, Phosphorylase_deoD B7LEN0 6414,6415, 6416 Purine_Nucleoside_(—) UniProtKB: 6417 6418 6419, 6420, 6421,Phosphorylase_deoD B7LXU6 6422, 6423, 6424 Purine_Nucleoside_(—)UniProtKB: 6425 6426 6427, 6428, 6429, Phosphorylase_deoD B7MNJ1 6430,6431, 6432 Purine_Nucleoside_(—) UniProtKB: 6433 6434 6435, 6436, 6437,Phosphorylase_deoD B7N2V8 6438, 6439, 6440 Purine_Nucleoside_(—)UniProtKB: 6441 6442 6443, 6444, 6445, Phosphorylase_deoD B7NH52 6446,6447, 6448 Purine_Nucleoside_(—) UniProtKB: 6449 6450 6451, 6452, 6453,Phosphorylase_deoD B7NW64 6454, 6455, 6456 Purine_Nucleoside_(—)UniProtKB: 6457 6458 6459, 6460, 6461, Phosphorylase_deoD B7UR12 6462,6463, 6464 Purine_Nucleoside_(—) UniProtKB: 6465 6466 6467, 6468, 6469,Phosphorylase_deoD C3SE47 6470, 6471, 6472 Purine_Nucleoside_(—)UniProtKB: 6473 6474 6475, 6476, 6477, Phosphorylase_deoD C4ZT66 6478,6479, 6480 Purine_Nucleoside_(—) UniProtKB: 6481 6482 6483, 6484, 6485,Phosphorylase_deoD C8TQD7 6486, 6487, 6488 Purine_Nucleoside_(—)UniProtKB: 6489 6490 6491, 6492, 6493, Phosphorylase_deoD C8U157 6494,6495, 6496 Purine_Nucleoside_(—) UniProtKB: 6497 6498 6499, 6500, 6501,Phosphorylase_deoD C8UN92 6502, 6503, 6504 Purine_Nucleoside_(—)UniProtKB: 6505 6506 6507, 6508, 6509, Phosphorylase_deoD D3GY24 6510,6511, 6512 Purine_Nucleoside_(—) UniProtKB: 6513 6514 6515, 6516, 6517,Phosphorylase_deoD D3QNE6 6518, 6519, 6520 Purine_Nucleoside_(—)UniProtKB: 6521 6522 6523, 6524, 6525, Phosphorylase_deoD D6I4N2 6526,6527, 6528 Purine_Nucleoside_(—) UniProtKB: 6529 6530 6531, 6532, 6533,Phosphorylase_deoD D6IHU2 6534, 6535, 6536 Purine_Nucleoside_(—)UniProtKB: 6537 6538 6539, 6540, 6541, Phosphorylase_deoD D6J6A4 6542,6543, 6544 Purine_Nucleoside_(—) UniProtKB: 6545 6546 6547, 6548, 6549,Phosphorylase_deoD E0J437 6550, 6551, 6552 Purine_Nucleoside_(—)UniProtKB: 6553 6554 6555, 6556, 6557, Phosphorylase_deoD E2QLE4 6558,6559, 6560 Purine_Nucleoside_(—) UniProtKB: 6561 6562 6563, 6564, 6565,Phosphorylase_deoD E3PFG7 6566, 6567, 6568 Purine_Nucleoside_(—)UniProtKB: 6569 6570 6571, 6572, 6573, Phosphorylase_deoD E8YEH0 6574,6575, 6576 Purine_Nucleoside_(—) UniProtKB: 6577 6578 6579, 6580, 6581,Phosphorylase_deoD F4NLK2 6582, 6583, 6584 Purine_Nucleoside_(—)UniProtKB: 6585 6586 6587, 6588, 6589, Phosphorylase_deoD F4SEX7 6590,6591, 6592 Purine_Nucleoside_(—) UniProtKB: 6593 6594 6595, 6596, 6597,Phosphorylase_deoD F4STB8 6598, 6599, 6600 Purine_Nucleoside_(—)UniProtKB: 6601 6602 6603, 6604, 6605, Phosphorylase_deoD F4T9F1 6606,6607, 6608 Purine_Nucleoside_(—) UniProtKB: 6609 6610 6611, 6612, 6613,Phosphorylase_deoD F4UXB7 6614, 6615, 6616 Purine_Nucleoside_(—)UniProtKB: 6617 6618 6619, 6620, 6621, Phosphorylase_deoD F4VN60 6622,6623, 6624 Purine_Nucleoside_(—) UniProtKB: 6625 6626 6627, 6628, 6629,Phosphorylase_deoD F4VQF8 6630, 6631, 6632 Purine_Nucleoside_(—)UniProtKB: 6633 6634 6635, 6636, 6637, Phosphorylase_deoD H9V0H4 6638,6639, 6640 Purine_Nucleoside_(—) UniProtKB: 6641 6642 6643, 6644, 6645,Phosphorylase_deoD J7QV83 6646, 6647, 6648 Purine_Nucleoside_(—)UniProtKB: 6649 6650 6651, 6652, 6653, Phosphorylase_deoD P0ABP8 6654,6655, 6656 Purine_Nucleoside_(—) UniProtKB: 6657 6658 6659, 6660, 6661,Phosphorylase_deoD Q0T8S9 6662, 6663, 6664 Purine_Nucleoside_(—)UniProtKB: 6665 6666 6667, 6668, 6669, Phosphorylase_deoD Q1R259 6670,6671, 6672 Purine_Nucleoside_(—) UniProtKB: 6673 6674 6675, 6676, 6677,Phosphorylase_deoD W8ZSE4 6678, 6679, 6680 Purine_Nucleoside_(—)UniProtKB: 6681 6682 6683, 6684, 6685, Phosphorylase_deoD X5FDR9 6686,6687, 6688 Thymidine_Kinase_TK UniProtKB: 6689 6690 6691, 6692, 6693,B2CPP5 6694, 6695, 6696 Thymidine_Kinase_TK UniProtKB: 6697 6698 6699,6700, 6701, B2CPP6 6702, 6703, 6704 Thymidine_Kinase_TK UniProtKB: 67056706 6707, 6708, 6709, B2CPP7 6710, 6711, 6712 Thymidine_Kinase_TKUniProtKB: 6713 6714 6715, 6716, 6717, B2CPP8 6718, 6719, 6720Thymidine_Kinase_TK UniProtKB: 6721 6722 6723, 6724, 6725, B2CPP9 6726,6727, 6728 Thymidine_Kinase_TK UniProtKB: 6729 6730 6731, 6732, 6733,B2CPQ0 6734, 6735, 6736 Thymidine_Kinase_TK UniProtKB: 6737 6738 6739,6740, 6741, B2CPQ2 6742, 6743, 6744 Thymidine_Kinase_TK UniProtKB: 67456746 6747, 6748, 6749, B2CPQ3 6750, 6751, 6752 Thymidine_Kinase_TKUniProtKB: 6753 6754 6755, 6756, 6757, B2CPQ4 6758, 6759, 6760Thymidine_Kinase_TK UniProtKB: 6761 6762 6763, 6764, 6765, B2CPQ5 6766,6767, 6768 Thymidine_Kinase_TK UniProtKB: 6769 6770 6771, 6772, 6773,O72346 6774, 6775, 6776 Thymidine_Kinase_TK UniProtKB: 6777 6778 6779,6780, 6781, P06478 6782, 6783, 6784 Thymidine_Kinase_TK UniProtKB: 67856786 6787, 6788, 6789, P08333 6790, 6791, 6792 Thymidine_Kinase_TKUniProtKB: 6793 6794 6795, 6796, 6797, Q9DLP2 6798, 6799, 6800Thymidine_Kinase_TK UniProtKB: 6801 6802 6803, 6804, 6805, Q9ENS0 6806,6807, 6808 Thymidine_Kinase_TK UniProtKB: 6809 6810 6811, 6812, 6813,Q9ENS1 6814, 6815, 6816 Thymidine_Kinase_TK UniProtKB: 6817 6818 6819,6820, 6821, Q9ENS2 6822, 6823, 6824 Thymidine_Kinase_TK UniProtKB: 68256826 6827, 6828, 6829, Q9ENS3 6830, 6831, 6832 Thymidine_Kinase_TKUniProtKB: 6833 6834 6835, 6836, 6837, Q9ENS4 6838, 6839, 6840Thymidine_Kinase_TK UniProtKB: 6841 6842 6843, 6844, 6845, Q9ENS5 6846,6847, 6848 Thymidine_Kinase_TK UniProtKB: 6849 6850 6851, 6852, 6853,Q9IYZ7 6854, 6855, 6856 Thymidine_Kinase_TK UniProtKB: 6857 6858 6859,6860, 6861, Q9IYZ9 6862, 6863, 6864 Thymidine_Kinase_TK UniProtKB: 68656866 6867, 6868, 6869, Q9IZ02 6870, 6871, 6872 Thymidine_Kinase_TKUniProtKB: 6873 6874 6875, 6876, 6877, Q9IZ03 6878, 6879, 6880Thymidine_Kinase_TK UniProtKB: 6881 6882 6883, 6884, 6885, Q9IZ07 6886,6887, 6888 Thymidine_Kinase_TK UniProtKB: 6889 6890 6891, 6892, 6893,Q9QNF7 6894, 6895, 6896

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one suicide geneproduct or a fragment or variant thereof, wherein the at least onecoding region comprises an RNA sequence being identical or at least 50%,60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNAsequences according to the SEQ ID Nos as disclosed in Table 4.

4. Immunogenic Proteins or Peptides

Preferably the RNA, preferably mRNA of the inventive RNA compositioncodes for at least one immunogenic protein or peptide, especially aprotein or peptide of a pathogen, preferably a viral pathogen, or afragment or variant thereof. By using RNA which codes for an immunogenicprotein or peptide which is preferably a pathogenic antigen it ispossible to utilize preexisting immunity against such antigens fortreatment of tumor and/or cancer diseases. The memory immune response istriggered and the immune system is strengthened for attacking tumorcells.

This embodiment of the invention is based on the recognition that inprinciple every organism with an immune system exhibits “memory immuneresponses” against certain foreign molecules (antigens), for exampleproteins, in particular viral or bacterial proteins. If an organism hasalready been infected at an earlier point in time with the antigen animmune response against e.g. the viral protein has already beentriggered by this infection. The immune system has a “memory” of thisresponse and stores it. As consequence of a reinfection with the antigenthe immune response is reactivated. Such reactivation may proceed byadministration of an RNA, preferably mRNA coding for the antigen,wherein the preferred intratumoral administration according to theinvention is especially effective. By reactivation of the memory immuneresponse against e.g. viral pathogens it is possible to destroy tumorcells effectively.

Preferred examples of immunogenic proteins or peptides for thisembodiment of the invention are proteins or peptides of widespreadpathogens, i.e. pathogens with which every organism, in particularmammals, preferably humans, has a high probability of being infected atleast once in his/her lifetime. These include, for example, anystructural or non-structural protein or peptide of:

-   -   influenza virus type A or B or any other orthomyxovirus        (influenza type C),    -   picornaviruses, such as rhinovirus or hepatitis A virus,    -   togaviruses, such as alphavirus or rubivirus, e.g. Sindbis,        Semliki-Forest or rubeolavirus (measles virus),    -   rubella virus (German measles virus),    -   coronaviruses, in particular subtypes HCV-229E or HCV-OC43,    -   rhabdoviruses, such as rabies virus,    -   paramyxoviruses, such as mumps virus,    -   reoviruses, such as group A, B or C rotavirus,    -   hepadnaviruses, such as hepatitis B virus,    -   papoviruses, such as human papillomaviruses (HPV) of any        serotype, especially from 1 to 75,    -   adenoviruses, in particular type 1 to 47,    -   herpesviruses, such as Herpes simplex virus 1, 2 or 3,    -   cytomegalovirus (CMV), preferably CMVpp65,    -   Epstein Barr virus (EBV),    -   vaccinia viruses and    -   the bacterium Chiamydophila pneumoniae (Chlamydia pneumoniae).

Further examples of preferred immunogenic proteins or peptides areproteins or peptides of pathogens which only seldom infect an organism.Nevertheless RNA coding for one or more of these proteins or peptidesmay be effective in the inventive approach. These proteins or peptideinclude, for example, any structural or non-structural protein orpeptide of:

-   -   Flaviviruses, such as dengue virus type 1 to 4, yellow fever        virus, West Nile virus, Japanese encephalitis virus    -   hepatitis C virus,    -   caliciviruses,    -   filoviruses, such as Ebola virus,    -   bornaviruses,    -   bunyaviruses, such as Rift Valley fever virus,    -   arenaviruses, such as LCMV (lymphocytic choriomeningitis virus)        or hemorrhagic fever viruses,    -   retroviruses, such as HIV and    -   parvoviruses.

Preferably the RNA of the inventive mRNA composition codes for influenzanucleoprotein (NP). It has been shown by the inventors that the use of acomposition containing mRNA coding for influenza nucleoprotein isespecially effective in reducing tumor size, when applied according tothe inventive approach. In this context an mRNA encoding an Influenzanucleoprotein according to SEQ ID NO. 6 is particularly preferred.

5. Cell Death Inducers and Apoptosis Inducers:

In the broadest sense, an apoptosis inducer or cell death inducer has tobe understood as a molecule inducing autophagy, cornification,excitotoxicity, necrosis, Wallerian degeneration, entosis, mitoticcatastrophe, necroptosis and pyroptosis (reviewed in Kroemer, G., et al.“Classification of cell death: recommendations of the NomenclatureCommittee on Cell Death 2009.” Cell Death & Differentiation 16.1 (2009):3-11.).

In a further preferred embodiment of the inventive RNA containingcomposition the RNA codes for at least one apoptosis inducer, preferablyan apoptosis inducer chosen from the group consisting of the Bcl-2family and tumor suppressor protein p53 and ligands of transmembranedeath receptors, especially the TNF (tumor necrosis factor) receptorgene superfamily, pro-apoptic receptor agonists and Beclin-1.

A particularly preferred apoptosis inducer in the context of the presentinvention is Beclin-1 (derived from the BECN1 gene). It is known in theart that Beclin-1 interacts with Bcl-2, BCL2L2, GOPC and MAP1LC3A toregulate autophagy and cell death.

Apoptosis provides an important barrier against cancer. However,specific mutations (e.g. mutation of the tumor suppressor gene p53)enable some tumor cells to escape apoptotic death and become moremalignant. By using an mRNA coding for at least one apoptosis inducer itis possible to reactivate apoptosis which is an important and effectivesystem of the organism to eliminate cancer cells.

Preferred examples of apoptosis inducers may be chosen from thefollowing list: Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, Blk, Cytochrome c,Caspases, especially Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase9, Death domain, especially of Fas, preferably FasL, TNFα, Apo2L/TRAIL,agonist of DR4 and/or DR5, Apo3L, DR4 agonistic antibody, DR5 agonisticantibody, protein kinase R (PKR) (preferably constitutive active PKR),Granzyme B.

Two signalling pathways initiate apoptosis: the intrinsic pathway actsthrough intracellular Bcl-2 proteins, the extrinsic pathway throughcell-surface pro-apoptotic receptors.

The intrinsic signaling pathway for programmed cell death involvesnon-receptor-mediated intracellular signals, inducing activities in themitochondria that initiate apoptosis. Stimuli for the intrinsic pathwayinclude viral infections or damage to the cell by toxins, free radicals,or radiation. Damage to the cellular DNA can also induce the activationof the intrinsic pathway for programmed cell death. These stimuli inducechanges in the inner mitochondrial membrane that result in the loss oftransmembrane potential, causing the release of pro-apoptotic proteinsinto the cytosol. Pro-apoptotic proteins activate caspases that mediatethe destruction of the cell through many pathways. These proteins alsotranslocate into the cellular nucleus, inducing DNA fragmentation, ahallmark of apoptosis. The regulation of pro-apoptotic events in themitochondria occurs through activity of members of the Bcl-2 family ofproteins and the tumor suppressor protein p53. Members of the Bcl-2family of proteins may be pro-apoptotic or anti-apoptotic. Theanti-apoptotic proteins are Bcl-2, Bcl-x, Bcl-xL, Bcl-XS, Bcl-w, andBAG. Pro-apoptotic proteins include Bcl-10, Bax, Bak, Bid, Bad, Bim,Bik, and Blk (Elmore, 2007. Toxicol Pathol. 35(4):495-516 (PMID:17562483)), which are especially preferred for the inventive approach.

The extrinsic signaling pathway leading to apoptosis involvestransmembrane death receptors that are members of the tumor necrosisfactor (TNF) receptor gene superfamily. Members of this receptor familybind to extrinsic ligands and transduce intracellular signals thatultimately result in the destruction of the cell. The most wellcharacterized ligands of these receptors to date are FasL, TNFα, Apo2L,and Apo3L. Corresponding receptors are FasR, TNFR1, DR3, and DR4/DR5.Molecules that stimulate the activity of these pro-apoptotic proteins oractivate these receptors are currently under evaluation for theirtherapeutic potential in the treatment of cancer, including hematologicmalignancies (Elmore, 2007. Toxicol Pathol. 35(4):495-516 (PMID:17562483)). These extrinsic ligands are further especially preferredexamples for use in the inventive approach.

New molecular insights have inspired the development of pro-apoptoticreceptor agonists (PARAs), including the recombinant human proteinapoptosis ligand 2/TNF-related apoptosis-inducing ligand (Apo2L/TRAIL).In addition, agonistic monoclonal antibodies to its signalling receptorsDR4 (TRAILR1) and DR5 (TRAILR2) are under development. Mapatumumab is anexample of a DR4 agonist antibody. Examples of DR5 agonistic antibodiesinclude Lexatumumab, Apomab, AMG655, CS-1008 and LBY-135 (Ashkenazi,2008. Nat. Rev. Drug Discov. 7(12):1001-12 (PMID: 18989337)).

The following table 5 summarizes some preferred apoptosis inducers.

TABLE 5 Apoptosis inducers Gene/Agent Example Intrinsic pathway Bcl-10Bax Bak Bid Bad Bim Bik Blk Cytochrome c Caspase 3, 6, 7, 8, 9 Extrinsicpathway FasL TNFα Apo2L/TRAIL Apo3L DR4 agonist antibody Mapatumumab DR5agonist antibody Lexatumumab, Apomab, AMG655, CS-1008, LBY-135 OtherGranzyme B

According to preferred embodiments in the context of the presentinvention apoptosis inducers may be selected from any apoptosis inducerselected from the group consisting of Apo2L/TRAIL; Apo3L; Bad; Bak; Bax;Bcl-10; Bid; Bik; Bim; Blk; Caspase_3; Caspase_6; Caspase_7; Caspase_8;Caspase_9; Cytochrome_c; FasL; Granzyme_B; TNF, preferably as disclosedin Table 6. Particularly preferred in this context are the RNA sequencesencoding an apoptosis inducer according to Table 6.

TABLE 6 Apoptosis inducers: RNA Optimized Protein Sequence RNA Sequencewild type Sequence Gene Name Protein Accession No. SEQ ID NO: SEQ ID NO:SEQ ID NO: Apo2L/TRAIL UniProtKB: P50591 6897 6898 6899, 6900, 6901,6902, 6903, 6904 Apo3L UniProtKB: O43508 6905 6906 6907, 6908, 6909,6910, 6911, 6912 Bad UniProtKB: A0A024R562 6913 6914 6915, 6916, 6917,6918, 6919, 6920 Bad UniProtKB: Q92934 6921 6922 6923, 6924, 6925, 6926,6927, 6928 Bak UniProtKB: Q16611 6929 6930 6931, 6932, 6933, 6934, 6935,6936 Bak UniProtKB: Q8NFF3 6937 6938 6939, 6940, 6941, 6942, 6943, 6944Bax UniProtKB: A0A0C4MVT1 6945 6946 6947, 6948, 6949, 6950, 6951, 6952Bax UniProtKB: A0A0C4MW46 6953 6954 6955, 6956, 6957, 6958, 6959, 6960Bax UniProtKB: A0A0C4MWS3 6961 6962 6963, 6964, 6965, 6966, 6967, 6968Bax UniProtKB: I6LPK7 6969 6970 6971, 6972, 6973, 6974, 6975, 6976 BaxUniProtKB: K4JQN1 6977 6978 6979, 6980, 6981, 6982, 6983, 6984 BaxUniProtKB: Q07812 6985 6986 6987, 6988, 6989, 6990, 6991, 6992 Bcl-10UniProtKB: O95999 6993 6994 6995, 6996, 6997, 6998, 6999, 7000 BidUniProtKB: A8ASI8 7001 7002 7003, 7004, 7005, 7006, 7007, 7008 BidUniProtKB: B2ZP78 7009 7010 7011, 7012, 7013, 7014, 7015, 7016 BidUniProtKB: B2ZP79 7017 7018 7019, 7020, 7021, 7022, 7023, 7024 BidUniProtKB: P55957 7025 7026 7027, 7028, 7029, 7030, 7031, 7032 BikUniProtKB: A0A024R4X6 7033 7034 7035, 7036, 7037, 7038, 7039, 7040 BikUniProtKB: Q13323 7041 7042 7043, 7044, 7045, 7046, 7047, 7048 BimUniProtKB: O43521 7049 7050 7051, 7052, 7053, 7054, 7055, 7056 BlkUniProtKB: P51451 7057 7058 7059, 7060, 7061, 7062, 7063, 7064 Caspase_3UniProtKB: P42574 7065 7066 7067, 7068, 7069, 7070, 7071, 7072 Caspase_6UniProtKB: P55212 7073 7074 7075, 7076, 7077, 7078, 7079, 7080 Caspase_7UniProtKB: P55210 7081 7082 7083, 7084, 7085, 7086, 7087, 7088 Caspase_8UniProtKB: B5BU46 7089 7090 7091, 7092, 7093, 7094, 7095, 7096 Caspase_8UniProtKB: B6CGU5 7097 7098 7099, 7100, 7101, 7102, 7103, 7104 Caspase_8UniProtKB: C3S3G0 7105 7106 7107, 7108, 7109, 7110, 7111, 7112 Caspase_8UniProtKB: Q14790 7113 7114 7115, 7116, 7117, 7118, 7119, 7120 Caspase_9UniProtKB: A0A024R8F1 7121 7122 7123, 7124, 7125, 7126, 7127, 7128Caspase_9 UniProtKB: A0A024R8I4 7129 7130 7131, 7132, 7133, 7134, 7135,7136 Caspase_9 UniProtKB: P55211 7137 7138 7139, 7140, 7141, 7142, 7143,7144 Caspase_9 UniProtKB: Q9H257 7145 7146 7147, 7148, 7149, 7150, 7151,7152 Cytochrome_c UniProtKB: A0A024R9B7 7153 7154 7155, 7156, 7157,7158, 7159, 7160 Cytochrome_c UniProtKB: A0A024RAP6 7161 7162 7163,7164, 7165, 7166, 7167, 7168 Cytochrome_c UniProtKB: A0A024RBN6 71697170 7171, 7172, 7173, 7174, 7175, 7176 Cytochrome_c UniProtKB:A0A024RBY9 7177 7178 7179, 7180, 7181, 7182, 7183, 7184 Cytochrome_cUniProtKB: B8XYC5 7185 7186 7187, 7188, 7189, 7190, 7191, 7192Cytochrome_c UniProtKB: G4XXL9 7193 7194 7195, 7196, 7197, 7198, 7199,7200 Cytochrome_c UniProtKB: H0UI06 7201 7202 7203, 7204, 7205, 7206,7207, 7208 Cytochrome_c UniProtKB: H6SG12 7209 7210 7211, 7212, 7213,7214, 7215, 7216 Cytochrome_c UniProtKB: H6SG13 7217 7218 7219, 7220,7221, 7222, 7223, 7224 Cytochrome_c UniProtKB: H6SG14 7225 7226 7227,7228, 7229, 7230, 7231, 7232 Cytochrome_c UniProtKB: H6SG15 7233 72347235, 7236, 7237, 7238, 7239, 7240 Cytochrome_c UniProtKB: O95101 72417242 7243, 7244, 7245, 7246, 7247, 7248 Cytochrome_c UniProtKB: P085747249 7250 7251, 7252, 7253, 7254, 7255, 7256 Cytochrome_c UniProtKB:P99999 7257 7258 7259, 7260, 7261, 7262, 7263, 7264 Cytochrome_cUniProtKB: Q496I0 7265 7266 7267, 7268, 7269, 7270, 7271, 7272Cytochrome_c UniProtKB: Q53XN1 7273 7274 7275, 7276, 7277, 7278, 7279,7280 Cytochrome_c UniProtKB: Q6FGA0 7281 7282 7283, 7284, 7285, 7286,7287, 7288 Cytochrome_c UniProtKB: Q6FGI7 7289 7290 7291, 7292, 7293,7294, 7295, 7296 Cytochrome_c UniProtKB: Q71U45 7297 7298 7299, 7300,7301, 7302, 7303, 7304 Cytochrome_c UniProtKB: Q86WV2 7305 7306 7307,7308, 7309, 7310, 7311, 7312 Cytochrome_c UniProtKB: Q9UEG9 7313 73147315, 7316, 7317, 7318, 7319, 7320 FasL UniProtKB: P48023 7321 73227323, 7324, 7325, 7326, 7327, 7328 Granzyme_B UniProtKB: J3KQ52 73297330 7331, 7332, 7333, 7334, 7335, 7336 Granzyme_B UniProtKB: Q67BC37337 7338 7339, 7340, 7341, 7342, 7343, 7344 Granzyme_B UniProtKB:Q6XGZ2 7345 7346 7347, 7348, 7349, 7350, 7351, 7352 Granzyme_BUniProtKB: Q6XGZ3 7353 7354 7355, 7356, 7357, 7358, 7359, 7360Granzyme_B UniProtKB: Q6XGZ4 7361 7362 7363, 7364, 7365, 7366, 7367,7368 TNF UniProtKB: P01375 7369 7370 7371, 7372, 7373, 7374, 7375, 7376TNF UniProtKB: Q5STB3 7377 7378 7379, 7380, 7381, 7382, 7383, 7384

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one apoptosisinducer or cell death inducer or a fragment or variant thereof, whereinthe at least one coding region comprises an RNA sequence being identicalor at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical tothe RNA sequences according to the SEQ ID Nos as disclosed in Table 6.

6. Angiogenesis Inhibitors

In a further preferred embodiment of the inventive RNA containingcomposition the at least one RNA, preferably mRNA codes for at least oneangiogenesis modulator or inhibitor, preferably an endogenousangiogenesis inhibitor or a fragment or variant thereof. Tumor growthand survival depend on angiogenesis to provide a path for delivery ofoxygen and nutrients to tumor cells. By using RNA coding for at leastone angiogenesis inhibitor according to the inventive approach it ispossible to block angiogenesis in a localized manner, namely within thetumor tissue, thereby providing an effective method for stopping tumorgrowth and decreasing tumor volume. Preferred examples of angiogenesisinhibitors according to the invention may be chosen from the followinglist: interferon alpha (IFN-α), (interferon beta) IFN-β, interferongamma (IFN-γ), CXCL9, CXCL10, interleukin 12 (IL-12), platelet factor 4(PF-4), tumor necrosis factor alpha (TNF-α), soluble fms-like tyrosinekinase 1 (sFLT-1), Fetal Liver Kinase 1 (FLK-1), Angiostatin,Endostatin, Vasostatin, Canstatin, Tumstatin, 16 kD prolacin fragment,tissue inhibitor of metalloproteinases 1 (TIMP-1), tissue inhibitor ofmetalloproteinases 2 (TIMP-2), tissue inhibitor of metalloproteinases 3(TIMP-3), thrombospondin 1 (TSP-1), thrombospondin 2 (TSP-2), Maspin,PEX, soluble Tyrosine-protein kinase receptor 1 (sTie1), solubleAngiopoietin-1 receptor 2 (sTie2), Angiopoietin-1, Angiopoietin-2,Antivascular endothelial growth factor receptor 2 (VEGFR2) antibody(e.g. Alacizumab, Ramucirumab), Anti-vascular endothelial growth factor(VEGF) antibody (e.g. Brolucizumab, Ranibizumab, Bevacizumab), andAnti-vascular endothelial growth factor receptor 1 (VEGFR1) antibody(e.g. Icrucumab).

Without this process of blood vessel recruitment, tumor growth islimited to 1 to 2 mm², the diffusion limit of oxygen. Already in 1971,Folkman proposed that tumor growth could be arrested by blockingangiogenesis (Folkman, 1972. N. Engl. J. Med. 285(21):1182-6).

Angiogenesis is a multistep process of new blood vessel formation frompreexisting vasculature that includes the activation, proliferation andmigration of endothelial cells (ECs), disruption of vascular basementmembranes, remodeling of the extracellular matrix of tissues, formationof vascular tubes and networks, recruitment of supporting cells,including smooth muscle cells and pericytes, and connection to thepre-existing vascular network.

Within a given microenvironment, the angiogenic response results from abalance between pro-angiogenic and anti-angiogenic factors, secretedboth by tumor cells and components of the stroma; the prevalence of theformer determines the “angiogenic switch”, resulting in the activationof angiogenesis followed by tumor outgrowth (Hanahan and Folkman, 1996.Cell 86(3):353-64).

Gene therapy based strategies of angiogenesis inhibition and especiallythe approach according to the present invention have several advantagescompared with conventional modalities of administration ofanti-angiogenic drugs. First of all, since effective suppression ofpathological angiogenesis may eventually require chronic treatment, thegene therapy strategy according to the invention is useful to achieveselective delivery to affected tissues and prolonged expression of thetherapeutic agents. Gene therapy in general also represents a method forcircumventing the production problems of many recombinant proteinsincluding their stability and solubility; adequate production ofanti-angiogenic factors by recombinant engineering methods has beensometimes problematic (e.g. for angiostatin) and may limit theirclinical application. Moreover gene transfer usage allows the correctfolding of proteic agents and their stability in vivo since they areassembled in their physiologic environment. A particularly attractivefeature of the inventive approach is the possibility of targeting genedelivery to selective tissues, namely tumor tissue, thus achievinglocalized gene expression and high regional drug concentrations withoutincreasing the systemic levels of the therapeutic agents and therebyresulting in an improved therapeutic index.

Angiogenesis inhibitors are heterogeneous in origin and potency, andtheir growing list includes proteolysis products of larger moleculeswith a different function, such as angiostatin, endostatin andvasostatin, modulators of vascular endothelial growth factor activity,such as soluble FLT-1 (sFLT-1), and some cytokines/chemokines withmarked anti-endothelial activity, such as IL-12, IFN-α, and CXCL10. Thefollowing table 8 (adapted from Persano et al., 2007. Mol. Aspects Med.28(1):87-114. PMID: 17306361) summarizes the preferred angiogenesisinhibitors which may be used in the inventive approach. According topreferred embodiments in the context of the present inventionangiogenesis inhibitors may be selected from any endogenous angiogenesisinhibitor selected from the group consisting of Angiopoietin-2;Angiostatin; Canstatin; CXCL10; CXCL4; CXCL9; Endostatin; FLK-1;IFNalpha; IFNB; IFNG; IL-12; PEX; PRL; SERPINB5; sFLT-1; sTie2; TIMP-1;TIMP-2; TIMP-3; TNF; TSP-1; TSP-2; Tumstatin; Vasostatin, preferably asdisclosed in Table 7. Particularly preferred in this context are the RNAsequences encoding an angiogenesis inhibitor according to Table 7.

TABLE 7 Endogenous angiogenesis inhibitors RNA Optimized ProteinSequence RNA Sequence wild type Sequence Gene Name Protein Accession No.SEQ ID NO: SEQ ID NO: SEQ ID NO: IFNalpha UniProtKB: G9JKF1 3953 39543955, 3956, 3957, 3958, 3959, 3960 IFNalpha UniProtKB: P01562 3961 39623963, 3964, 3965, 3966, 3967, 3968 IFNalpha UniProtKB: P01563 3969 39703971, 3972, 3973, 3974, 3975, 3976 IFNalpha UniProtKB: P01566 3977 39783979, 3980, 3981, 3982, 3983, 3984 IFNalpha UniProtKB: P01567 3985 39863987, 3988, 3989, 3990, 3991, 3992 IFNalpha UniProtKB: P01568 3993 39943995, 3996, 3997, 3998, 3999, 4000 IFNalpha UniProtKB: P01569 4001 40024003, 4004, 4005, 4006, 4007, 4008 IFNalpha UniProtKB: P01570 4009 40104011, 4012, 4013, 4014, 4015, 4016 IFNalpha UniProtKB: P01571 4017 40184019, 4020, 4021, 4022, 4023, 4024 IFNalpha UniProtKB: P05013 4025 40264027, 4028, 4029, 4030, 4031, 4032 IFNalpha UniProtKB: P05014 4033 40344035, 4036, 4037, 4038, 4039, 4040 IFNalpha UniProtKB: P05015 4041 40424043, 4044, 4045, 4046, 4047, 4048 IFNalpha UniProtKB: P32881 4049 40504051, 4052, 4053, 4054, 4055, 4056 IFNalpha UniProtKB: Q14618 4057 40584059, 4060, 4061, 4062, 4063, 4064 IFNalpha UniProtKB: Q86UP4 4065 40664067, 4068, 4069, 4070, 4071, 4072 IFNB UniProtKB: P01574 4073 40744075, 4076, 4077, 4078, 4079, 4080 IFNB UniProtKB: Q15943 4081 40824083, 4084, 4085, 4086, 4087, 4088 IFNG UniProtKB: P01579 4089 40904091, 4092, 4093, 4094, 4095, 4096 IFNG UniProtKB: Q14609 4097 40984099, 4100, 4101, 4102, 4103, 4104 IFNG UniProtKB: Q14610 4105 41064107, 4108, 4109, 4110, 4111, 4112 IFNG UniProtKB: Q14611 4113 41144115, 4116, 4117, 4118, 4119, 4120 IFNG UniProtKB: Q14612 4121 41224123, 4124, 4125, 4126, 4127, 4128 IFNG UniProtKB: Q14613 4129 41304131, 4132, 4133, 4134, 4135, 4136 IFNG UniProtKB: Q14614 4137 41384139, 4140, 4141, 4142, 4143, 4144 IFNG UniProtKB: Q14615 4145 41464147, 4148, 4149, 4150, 4151, 4152 IFNG UniProtKB: Q8NHY9 4153 41544155, 4156, 4157, 4158, 4159, 4160 IL-12 UniProtKB: P29460 4193 41944195, 4196, 4197, 4198, 4199, 4200 CXCL10 UniProtKB: A0A024RDA4 51295130 5131, 5132, 5133, 5134, 5135, 5136 CXCL10 UniProtKB: P02778 51375138 5139, 5140, 5141, 5142, 5143, 5144 CXCL4 UniProtKB: P02776 52255226 5227, 5228, 5229, 5230, 5231, 5232 CXCL9 UniProtKB: L8E8X0 52735274 5275, 5276, 5277, 5278, 5279, 5280 CXCL9 UniProtKB: Q07325 52815282 5283, 5284, 5285, 5286, 5287, 5288 TNF UniProtKB: P01375 7369 73707371, 7372, 7373, 7374, 7375, 7376 TNF UniProtKB: Q5STB3 7377 7378 7379,7380, 7381, 7382, 7383, 7384 Angiopoietin-2 UniProtKB: B2R6E3 7385 73867387, 7388, 7389, 7390, 7391, 7392 Angiopoietin-2 UniProtKB: O15123 73937394 7395, 7396, 7397, 7398, 7399, 7400 Angiostatin UniProtKB:A0A0F7G8J1 7401 7402 7403, 7404, 7405, 7406, 7407, 7408 AngiostatinUniProtKB: P00747 7409 7410 7411, 7412, 7413, 7414, 7415, 7416Angiostatin UniProtKB: Q5TEH5 7417 7418 7419, 7420, 7421, 7422, 7423,7424 Canstatin UniProtKB: P08572 7425 7426 7427, 7428, 7429, 7430, 7431,7432 Endostatin Homo_sapiens 7433 7434 7435, 7436, 7437, 7438, 7439,7440 FLK-1 UniProtKB: P35968 7441 7442 7443, 7444, 7445, 7446, 7447,7448 PEX UniProtKB: P78562 7449 7450 7451, 7452, 7453, 7454, 7455, 7456PRL UniProtKB: P01236 7457 7458 7459, 7460, 7461, 7462, 7463, 7464SERPINB5 UniProtKB: P36952 7465 7466 7467, 7468, 7469, 7470, 7471, 7472sFLT-1 UniProtKB: H9N1E7 7473 7474 7475, 7476, 7477, 7478, 7479, 7480sFLT-1 UniProtKB: H9N1E8 7481 7482 7483, 7484, 7485, 7486, 7487, 7488sFLT-1 UniProtKB: L7RSL3 7489 7490 7491, 7492, 7493, 7494, 7495, 7496sFLT-1 UniProtKB: P17948 7497 7498 7499, 7500, 7501, 7502, 7503, 7504sTie2 UniProtKB: B5A953 7505 7506 7507, 7508, 7509, 7510, 7511, 7512TIMP-1 UniProtKB: P01033 7513 7514 7515, 7516, 7517, 7518, 7519, 7520TIMP-2 UniProtKB: P16035 7521 7522 7523, 7524, 7525, 7526, 7527, 7528TIMP-3 UniProtKB: P35625 7529 7530 7531, 7532, 7533, 7534, 7535, 7536TSP-1 UniProtKB: P07996 7537 7538 7539, 7540, 7541, 7542, 7543, 7544TSP-2 UniProtKB: P35442 7545 7546 7547, 7548, 7549, 7550, 7551, 7552Tumstatin UniProtKB: Q01955 7553 7554 7555, 7556, 7557, 7558, 7559, 7560Vasostatin UniProtKB: P10645 7561 7562 7563, 7564, 7565, 7566, 7567,7568

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one angiogenesisinhibitor or a fragment or variant thereof, wherein the at least onecoding region comprises an RNA sequence being identical or at least 50%,60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNAsequences according to the SEQ ID Nos as disclosed in Table 7.

7. Heat Shock Proteins

In a further preferred embodiment of the inventive RNA containingcomposition the RNA codes for at least one heat shock protein (HSP) or afragment or variant thereof. Preferably, the heat shock protein may bechosen from the following list: HSP27, HSP47 (serpin H1), HSP60, HSP70,HSC70, GRP78 (BiP), HSP90, HSP110, GRP94 (gp96), GRP170 (ORP150),PDI/PDIA, CRT/CALR.

As reviewed by Graner et al. (Graner M W, Lillehei K O, Katsanis E.Endoplasmic reticulum chaperones and their roles in the immunogenicityof cancer vaccines. Front Oncol. 2015 Jan. 6; 4:379. doi:10.3389/fonc.2014.00379) heat shock proteins play essential cellularhousekeeping functions and are indispensible during protein synthesis,folding and transport across intracellular membranes as well as proteindegradation. HSPs belong to a multiprotein family of chaperons whichconsists of, but is not limited to, HSP27, HSP47 (serpin H1), HSP60,HSP70, HSC70, GRP78 (BiP), HSP90, HSP110, GRP94 (gp96), GRP170 (ORP150),PDI/PDIA, CRT/CALR. In addition to the intracellular functions aschaperons, HSPs have been shown to play an important extracellular roleas simulators of the immune responses particularly in tumor settings.Various literature reports demonstrated that tumor-derived HSP-peptidecomplexes induce anti-tumor immune responses very efficiently. Themolecular mechanism of these observations has been elucidated. HSPs aschaperons have the capacity to bind denatured peptides including theantigenic ones and those complexes are internalized by antigenpresenting cells (APCs) which eventually leads to antigen presentationand induction of immunity. In addition to their chaperon function, HSPshave been shown to trigger danger signals in the tumor microenvironmentand thus stimulate macrophages and dendritic cells (DCs) to produceproinflammatory cytokines and enhance the induced immune responses.

According to preferred embodiments in the context of the presentinvention heat shock proteins may be selected from any heat shockprotein selected from the group consisting of calreticulin;GRP170_(ORP150); GRP78_(BiP); GRP94_(gp96); HSC70; HSP110; HSP27;HSP47_(serpin_H1); HSP60; HSP70; HSP90; PDI/PDIA, preferably asdisclosed in Table 8. Particularly preferred in this context are the RNAsequences encoding a heat shock protein according to Table 8.

TABLE 8 Heat shock proteins RNA Optimized Protein Sequence RNA Sequencewild type Sequence Gene Name Protein Accession No. SEQ ID NO: SEQ ID NO:SEQ ID NO: calreticulin UniProtKB: B4DHR1 7569 7570 7571, 7572, 7573,7574, 7575, 7576 calreticulin UniProtKB: B4E2Y9 7577 7578 7579, 7580,7581, 7582, 7583, 7584 calreticulin UniProtKB: P27797 7585 7586 7587,7588, 7589, 7590, 7591, 7592 calreticulin UniProtKB: Q96L12 7593 75947595, 7596, 7597, 7598, 7599, 7600 GRP170_(ORP150) UniProtKB: Q9Y4L17601 7602 7603, 7604, 7605, 7606, 7607, 7608 GRP78_(BiP) UniProtKB:P11021 7609 7610 7611, 7612, 7613, 7614, 7615, 7616 GRP94_(gp96)UniProtKB: P14625 7617 7618 7619, 7620, 7621, 7622, 7623, 7624 HSC70UniProtKB: P11142 7625 7626 7627, 7628, 7629, 7630, 7631, 7632 HSP110UniProtKB: Q92598 7633 7634 7635, 7636, 7637, 7638, 7639, 7640 HSP27UniProtKB: P04792 7641 7642 7643, 7644, 7645, 7646, 7647, 7648HSP47_(serpin_H1) UniProtKB: P50454 7649 7650 7651, 7652, 7653, 7654,7655, 7656 HSP60 UniProtKB: A0A024R3X4 7657 7658 7659, 7660, 7661, 7662,7663, 7664 HSP60 UniProtKB: B3GQS7 7665 7666 7667, 7668, 7669, 7670,7671, 7672 HSP60 UniProtKB: P10809 7673 7674 7675, 7676, 7677, 7678,7679, 7680 HSP60 UniProtKB: Q0VDF9 7681 7682 7683, 7684, 7685, 7686,7687, 7688 HSP70 UniProtKB: P38646 7689 7690 7691, 7692, 7693, 7694,7695, 7696 HSP90 UniProtKB: P07900 7697 7698 7699, 7700, 7701, 7702,7703, 7704 HSP90 UniProtKB: P08238 7705 7706 7707, 7708, 7709, 7710,7711, 7712 PDI/PDIA UniProtKB: P07237 7713 7714 7715, 7716, 7717, 7718,7719, 7720 PDI/PDIA UniProtKB: Q6YPB00 7721 7722 7723, 7724, 7725, 7726,7727, 7728 PDI/PDIA UniProtKB: Q71S60 7729 7730 7731, 7732, 7733, 7734,7735, 7736

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one heat shockprotein or a fragment or variant thereof, wherein the at least onecoding region comprises an RNA sequence being identical or at least 50%,60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNAsequences according to the SEQ ID Nos as disclosed in Table 8.

8. Tumor Antigens

In a further preferred embodiment of the inventive RNA containingcomposition the composition may contain RNA, preferably mRNA which codesfor at least one tumor antigen or a fragment or variant thereof, whichare used for vaccination to induce an adaptive immune response accordingto the invention.

In this context tumor antigens are particularly preferred to be encodedby RNA, preferably mRNA comprised in the inventive RNA composition. Itis particularly preferred that the inventive RNA composition comprisesat least one RNA encoding at least one tumor antigen or a fragment orvariant thereof.

Tumor antigens are preferably located on the surface of the (tumor)cell. Tumor antigens may also be selected from proteins, which areoverexpressed in tumor cells compared to a normal cell. Furthermore,tumor antigens also includes antigens expressed in cells which are(were) not themselves (or originally not themselves) degenerated but areassociated with the supposed tumor. Antigens which are connected withtumor-supplying vessels or (re)formation thereof, in particular thoseantigens which are associated with neovascularization, e.g. growthfactors, such as VEGF, bFGF etc., are also included herein. Antigensconnected with a tumor furthermore include antigens from cells ortissues, typically embedding the tumor. Further, some substances(usually proteins or peptides) are expressed in patients suffering(knowingly or not-knowingly) from a cancer disease and they occur inincreased concentrations in the body fluids of said patients. Thesesubstances are also referred to as “tumor antigens”, however they arenot antigens in the stringent meaning of an immune response inducingsubstance. The class of tumor antigens can be divided further intotumor-specific antigens (TSAs) and tumor-associated-antigens (TAAs).TSAs can only be presented by tumor cells and never by normal “healthy”cells. They typically result from a tumor specific mutation. TAAs, whichare more common, are usually presented by both tumor and healthy cells.These antigens are recognized and the antigen-presenting cell can bedestroyed by cytotoxic T cells. Additionally, tumor antigens can alsooccur on the surface of the tumor in the form of, e.g., a mutatedreceptor. In this case, they can be recognized by antibodies.

Further, tumor associated antigens may be classified as tissue-specificantigens, also called melanocyte-specific antigens, cancer-testisantigens and tumor-specific antigens. Cancer-testis antigens aretypically understood to be peptides or proteins of germ-line associatedgenes which may be activated in a wide variety of tumors. Humancancer-testis antigens may be further subdivided into antigens which areencoded on the X chromosome, so-called CT-X antigens, and those antigenswhich are not encoded on the X chromosome, the so-called (non-X CTantigens). Cancer-testis antigens which are encoded on the X-chromosomecomprises, for example, the family of melanoma antigen genes, theso-called MAGE-family. The genes of the MAGE-family may be characterisedby a shared MAGE homology domain (MHD). Each of these antigens, i.e.melanocyte-specific antigens, cancer-testis antigens and tumor-specificantigens, may elicit autologous cellular and humoral immune response.Accordingly, the tumor antigen encoded by the inventive nucleic acidsequence is preferably a melanocyte-specific antigen, a cancer-testisantigen or a tumor-specific antigens, preferably it may be a CT-Xantigen, a non-X CT-antigens, a binding partner for a CT-X antigen or abinding partner for a non-X CT-antigen or a tumor-specific antigen, morepreferably a CT-X antigen, a binding partner for a non-X CT-antigen or atumor-specific antigen.

Particular preferred tumor antigens are selected from the listconsisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m,alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1,BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsinB, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55,CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66,COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m,EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2,FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin,Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin classI/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE,PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1,survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP,TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1,TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, andWT1. Such tumor antigens preferably may be selected from the groupconsisting of p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1, MAGE-A3,Mesothelin, MUC-1, GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA, STEAP-1,VEGF, VEGFR1, VEGFR2, Ras, CEA or WT1, and more preferably from PAP,MAGE-A3, WT1, and MUC-1. Such tumor antigens preferably may be selectedfrom the group consisting of MAGE-A1 (e.g. MAGE-A1 according toaccession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6according to accession number NM_005363), MAGE-C1, MAGE-C2, melan-A(e.g. melan-A according to accession number NM_005511), GP100 (e.g.GP100 according to accession number M77348), tyrosinase (e.g. tyrosinaseaccording to accession number NM_000372), surviving (e.g. survivinaccording to accession number AF077350), CEA (e.g. CEA according toaccession number NM_004363), Her-2/neu (e.g. Her-2/neu according toaccession number M11730), WT1 (e.g. WT1 according to accession numberNM_000378), PRAME (e.g. PRAME according to accession number NM_006115),EGFRI (epidermal growth factor receptor 1) (e.g. EGFRI (epidermal growthfactor receptor 1) according to accession number AF288738), MUC1,mucin-1 (e.g. mucin-1 according to accession number NM_002456), SEC61G(e.g. SEC61G according to accession number NM_014302), hTERT (e.g. hTERTaccession number NM_198253), 5T4 (e.g. 5T4 according to accession numberNM_006670), TRP-2 (e.g. TRP-2 according to accession number NM_001922),STEAP1, PCA, PSA, PSMA, etc.

According to preferred embodiments in the context of the presentinvention tumor antigens may be selected from any tumor antigen selectedfrom the group consisting of 1A01_HLA-A/m; 1A02; 5T4; ACRBP; AFP; AKAP4;alpha-actinin-_4/m; alpha-methylacyl-coenzyme_A_racemase; ANDR; ART-4;ARTC1/m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr/abl;beta-catenin/m; BING-4; BIRC7; BRCA1/m; BY55; calreticulin; CAMEL;CASPA; Caspase_8; cathepsin_B; cathepsin_L; CD1A; CD1B; CD1C; CD1D;CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44_Isoform_1;CD44_Isoform_6; CD52; CD55; CD56; CD80; CD86; CD8A; CDC27/m; CDE30;CDK4/m; CDKN2A/m; CEA; CEAM6; CH3L2; CLCA2; CML28; CML66; COA-1/m;coactosin-like_protein; collagen_XXIII; COX-2; CP1B1; CSAG2; CT-_9/BRD6;CT45A1; CT55; CTAG2_Isoform_LAGE-1A; CTAG2_Isoform_LAGE-1B; CTCFL; Cten;cyclin_B1; cyclin_D1; cyp-B; DAM-10; DEP1A; E7; EF1A2; EFTUD2/m; EGFR;EGLN3; ELF2/m; EMMPRIN; EpCam; EphA2; EphA3; ErbB3; ERBB4; ERG; ETV6;EWS; EZH2; FABP7; FCGR3A_Version_1; FCGR3A_Version_2; FGF5; FGFR2;fibronectin; FOS; FOXP3; FUT1; G250; GAGE-1; GAGE-2; GAGE-3; GAGE-4;GAGE-5; GAGE-6; GAGE7b; GAGE-8_(GAGE-2D); GASR; GnT-V; GPC3; GPNMB/m;GRM3; HAGE; hepsin; Her2/neu; HLA-A2/m; homeobox_NKX3.1; HOM-TES-85;HPG1; HS71A; HS71B; HST-2; hTERT; iCE; IF2B3; IL-10; IL-13Ra2; IL2-RA;IL2-RB; IL2-RG; IL-5; IMP3; ITA5; ITB1; ITB6; kallikrein-2;kallikrein-4; KI20A; KIAA0205; KIF2C; KK-LC-1; LDLR; LGMN; LIRB2; LY6K;MAGA5; MAGA8; MAGAB; MAGE-_B1; MAGE-_E1; MAGE-A1; MAGE-A10; MAGE-A12;MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-B10; MAGE-B16;MAGE-B17; MAGE-B2; MAGE-B3; MAGE-B4; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2;MAGE-C3; MAGE-D1; MAGE-D2; MAGE-D4; MAGE-E1_(MAGE1); MAGE-E2; MAGE-F1;MAGE-H1; MAGEL2; mammaglobin_A; MART-1/melan-A; MART-2; MC1_R; M-CSF;mesothelin; MITF; MMP1_1; MMP7; MUC-1; MUM-1/m; MUM-2/m; MYO1A; MYO1B;MYO1C; MYO1D; MYO1E; MYO1F; MYO1G; MYO1H; NA17; NA88-A; Neo-PAP; NFYC/m;NGEP; N-myc; NPM; NRCAM; NSE; NUF2; NY-ESO-1; OA1; OGT; OS-9;osteocalcin; osteopontin; p53; PAGE-4; PAI-1; PAI-2; PAP; PATE; PAX3;PAX5; PD1L1; PDCD1; PDEF; PECA1; PGCB; PGFRB; Pim-1_-Kinase; Pin-1;PLAC1; PMEL; PML; POTE; POTEF; PRAME; PRDX5/m; PRM2; prostein;proteinase-3; PSA; PSB9; PSCA; PSGR; PSM; PTPRC; RAB8A; RAGE-1; RARA;RASH; RASK; RASN; RGS5; RHAMM/CD168; RHOC; RSSA; RU1; RU2; RUNX1; S-100;SAGE; SART-_1; SART-2; SART-3; SEPR; SERPINB5; SIA7F; SIA8A; SIAT9;SIRT2/m; SOX10; SP17; SPNXA; SPXN3; SSX-1; SSX-2; SSX3; SSX-4; ST1A1;STAG2; STAMP-1; STEAP-1; survivin; Survivin-2B; SYCP1; SYT-SSX-1;SYT-SSX-2; TARP; TCRg; TF2AA; TGFbeta1; TGFR2; TGM-4; TIE2; TKTL1;TPI/m; TRGV11; TRGV9; TRPC1; TRP-p8; TSG10; TSPY1; TVC_(TRGV3); TX101;tyrosinase; TYRP1; TYRP2; UPA; VEGFR1; WT1; XAGE1, preferably asdisclosed in Table 9. Particularly preferred in this context are the RNAsequences encoding a tumor antigen according to Table 9.

TABLE 9 Tumor antigens RNA Optimized Protein Sequence RNA Sequence wildtype Sequence Gene Name Protein Accession No. SEQ ID NO: SEQ ID NO: SEQID NO: 1A01_HLA-A/m UniProtKB: P30443 398 399 400, 401, 402, 403, 4041A02 UniProtKB: P01892 405 406 407, 408, 409, 410, 411 5T4 UniProtKB:Q13641 412 413 414, 415, 416, 417, 418 ACRBP UniProtKB: Q8NEB7 419 420421, 422, 423, 424, 425 AFP UniProtKB: P02771 426 427 428, 429, 430,431, 432 AKAP4 UniProtKB: Q5JQC9 433 434 435, 436, 437, 438, 439alpha-actinin-_4/m UniProtKB: B4DSX0 440 441 442, 443, 444, 445, 446alpha-actinin-_4/m UniProtKB: B4E337 447 448 449, 450, 451, 452, 453alpha-actinin-_4/m UniProtKB: O43707 454 455 456, 457, 458, 459, 460alpha-methylacyl- UniProtKB: A0A024RE16 461 462 463, 464, 465, 466, 467coenzyme_A_racemase alpha-methylacyl- UniProtKB: A8KAC3 468 469 470,471, 472, 473, 474 coenzyme_A_racemase ANDR UniProtKB: P10275 475 476477, 478, 479, 480, 481 ART-4 UniProtKB: Q9ULX3 482 483 484, 485, 486,487, 488 ARTC1/m UniProtKB: P52961 489 490 491, 492, 493, 494, 495 AURKBUniProtKB: Q96GD4 496 497 498, 499, 500, 501, 502 B2MG UniProtKB: P61769503 504 505, 506, 507, 508, 509 B3GN5 UniProtKB: Q9BYG0 510 511 512,513, 514, 515, 516 B4GN1 UniProtKB: Q00973 517 518 519, 520, 521, 522,523 B7H4 UniProtKB: Q7Z7D3 524 525 526, 527, 528, 529, 530 BAGE-1UniProtKB: Q13072 531 532 533, 534, 535, 536, 537 BASI UniProtKB: P35613538 539 540, 541, 542, 543, 544 BCL-2 UniProtKB: A9QXG9 545 546 547,548, 549, 550, 551 bcr/abl UniProtKB: A9UEZ4 552 553 554, 555, 556, 557,558 bcr/abl UniProtKB: A9UEZ7 559 560 561, 562, 563, 564, 565 bcr/ablUniProtKB: A9UEZ8 566 567 568, 569, 570, 571, 572 bcr/abl UniProtKB:A9UEZ9 573 574 575, 576, 577, 578, 579 bcr/abl UniProtKB: A9UF00 580 581582, 583, 584, 585, 586 bcr/abl UniProtKB: A9UF01 587 588 589, 590, 591,592, 593 bcr/abl UniProtKB: A9UF03 594 595 596, 597, 598, 599, 600bcr/abl UniProtKB: A9UF04 601 602 603, 604, 605, 606, 607 bcr/ablUniProtKB: A9UF05 608 609 610, 611, 612, 613, 614 bcr/abl UniProtKB:A9UF06 615 616 617, 618, 619, 620, 621 bcr/abl UniProtKB: A9UF08 622 623624, 625, 626, 627, 628 beta-catenin/m UniProtKB: P35222 629 630 631,632, 633, 634, 635 beta-catenin/m UniProtKB: Q8WYA6 636 637 638, 639,640, 641, 642 BING-4 UniProtKB: O15213 643 644 645, 646, 647, 648, 649BIRC7 UniProtKB: Q96CA5 650 651 652, 653, 654, 655, 656 BRCA1/mUniProtKB: A0A024R1V0 657 658 659, 660, 661, 662, 663 BRCA1/m UniProtKB:A0A024R1V7 664 665 666, 667, 668, 669, 670 BRCA1/m UniProtKB: A0A024R1Z8671 672 673, 674, 675, 676, 677 BRCA1/m UniProtKB: A0A068BFX7 678 679680, 681, 682, 683, 684 BRCA1/m UniProtKB: C6YB45 685 686 687, 688, 689,690, 691 BRCA1/m UniProtKB: C6YB47 692 693 694, 695, 696, 697, 698BRCA1/m UniProtKB: G3XAC3 699 700 701, 702, 703, 704, 705 BY55UniProtKB: O95971 706 707 708, 709, 710, 711, 712 CAMEL UniProtKB:O95987 713 714 715, 716, 717, 718, 719 CASPA UniProtKB: Q92851-4 720 721722, 723, 724, 725, 726 cathepsin_B UniProtKB: A0A024R374 727 728 729,730, 731, 732, 733 cathepsin_B UniProtKB: P07858 734 735 736, 737, 738,739, 740 cathepsin_L UniProtKB: A0A024R276 741 742 743, 744, 745, 746,747 cathepsin_L UniProtKB: P07711 748 749 750, 751, 752, 753, 754cathepsin_L UniProtKB: Q9HBQ7 755 756 757, 758, 759, 760, 761 CD1AUniProtKB: P06126 762 763 764, 765, 766, 767, 768 CD1B UniProtKB: P29016769 770 771, 772, 773, 774, 775 CD1C UniProtKB: P29017 776 777 778, 779,780, 781, 782 CD1D UniProtKB: P15813 783 784 785, 786, 787, 788, 789CD1E UniProtKB: P15812 790 791 792, 793, 794, 795, 796 CD20 UniProtKB:P11836 797 798 799, 800, 801, 802, 803 CD22 UniProtKB: O60926 804 805806, 807, 808, 809, 810 CD22 UniProtKB: P20273 811 812 813, 814, 815,816, 817 CD22 UniProtKB: Q0EAF5 818 819 820, 821, 822, 823, 824 CD276UniProtKB: Q5ZPR3 825 826 827, 828, 829, 830, 831 CD33 UniProtKB: B4DF51832 833 834, 835, 836, 837, 838 CD33 UniProtKB: P20138 839 840 841, 842,843, 844, 845 CD33 UniProtKB: Q546G0 846 847 848, 849, 850, 851, 852CD3E UniProtKB: P07766 853 854 855, 856, 857, 858, 859 CD3Z UniProtKB:P20963 860 861 862, 863, 864, 865, 866 CD44_Isoform_1 UniProtKB: P16070867 868 869, 870, 871, 872, 873 CD44_Isoform_6 UniProtKB: P16070-6 874875 876, 877, 878, 879, 880 CD4 UniProtKB: P01730 881 882 883, 884, 885,886, 887 CD52 UniProtKB: P31358 888 889 890, 891, 892, 893, 894 CD52UniProtKB: Q6IBD0 895 896 897, 898, 899, 900, 901 CD52 UniProtKB: V9HWN9902 903 904, 905, 906, 907, 908 CD55 UniProtKB: B1AP15 909 910 911, 912,913, 914, 915 CD55 UniProtKB: D3DT85 916 917 918, 919, 920, 921, 922CD55 UniProtKB: D3DT86 923 924 925, 926, 927, 928, 929 CD55 UniProtKB:P08174 930 931 932, 933, 934, 935, 936 CD56 UniProtKB: P13591 937 938939, 940, 941, 942, 943 CD80 UniProtKB: A0N0P2 944 945 946, 947, 948,949, 950 CD80 UniProtKB: P33681 951 952 953, 954, 955, 956, 957 CD86UniProtKB: P42081 958 959 960, 961, 962, 963, 964 CD8A UniProtKB: P01732965 966 967, 968, 969, 970, 971 CDC27/m UniProtKB: G5EA36 972 973 974,975, 976, 977, 978 CDC27/m UniProtKB: P30260 979 980 981, 982, 983, 984,985 CDE30 UniProtKB: P28908 986 987 988, 989, 990, 991, 992 CDK4/mUniProtKB: A0A024RBB6 993 994 995, 996, 997, 998, 999 CDK4/m UniProtKB:P11802 1000 1001 1002, 1003, 1004, 1005, 1006 CDK4/m UniProtKB: Q6LC831007 1008 1009, 1010, 1011, 1012, 1013 CDK4/m UniProtKB: Q96BE9 10141015 1016, 1017, 1018, 1019, 1020 CDKN2A/m UniProtKB: D1LYX3 1021 10221023, 1024, 1025, 1026, 1027 CDKN2A/m UniProtKB: G3XAG3 1028 1029 1030,1031, 1032, 1033, 1034 CDKN2A/m UniProtKB: K7PML8 1035 1036 1037, 1038,1039, 1040, 1041 CDKN2A/m UniProtKB: L8E941 1042 1043 1044, 1045, 1046,1047, 1048 CDKN2A/m UniProtKB: Q8N726 1049 1050 1051, 1052, 1053, 1054,1055 CEA RefSeq: NP_004354 1056 1057 1058, 1059, 1060, 1061, 1062 CEAM6UniProtKB: P40199 1063 1064 1065, 1066, 1067, 1068, 1069 CH3L2UniProtKB: Q15782 1070 1071 1072, 1073, 1074, 1075, 1076 CLCA2UniProtKB: Q9UQC9 1077 1078 1079, 1080, 1081, 1082, 1083 CML28UniProtKB: Q9NQT4 1084 1085 1086, 1087, 1088, 1089, 1090 CML66UniProtKB: Q96RS6 1091 1092 1093, 1094, 1095, 1096, 1097 COA-1/mUniProtKB: Q5T124 1098 1099 1100, 1101, 1102, 1103, 1104coactosin-like_protein UniProtKB: Q14019 1105 1106 1107, 1108, 1109,1110, 1111 collagen_XXIII UniProtKB: L8EAS4 1112 1113 1114, 1115, 1116,1117, 1118 collagen_XXIII UniProtKB: Q86Y22 1119 1120 1121, 1122, 1123,1124, 1125 COX-2 UniProtKB: Q6ZYK7 1126 1127 1128, 1129, 1130, 1131,1132 CP1B1 UniProtKB: Q16678 1133 1134 1135, 1136, 1137, 1138, 1139CSAG2 UniProtKB: Q9Y5P2-2 1140 1141 1142, 1143, 1144, 1145, 1146 CSAG2UniProtKB: Q9Y5P2 1147 1148 1149, 1150, 1151, 1152, 1153 CT45A1UniProtKB: Q5HYN5 1154 1155 1156, 1157, 1158, 1159, 1160 CT55 UniProtKB:Q8WUE5 1161 1162 1163, 1164, 1165, 1166, 1167 CT-_9/BRD6 UniProtKB:Q58F21 1168 1169 1170, 1171, 1172, 1173, 1174 CTAG2_Isoform_LAGE-UniProtKB: O75638-2 1175 1176 1177, 1178, 1179, 1A 1180, 1181CTAG2_Isoform_LAGE- UniProtKB: O75638 1182 1183 1184, 1185, 1186, 1B1187, 1188 CTCFL UniProtKB: Q8NI51 1189 1190 1191, 1192, 1193, 1194,1195 Cten UniProtKB: Q8IZW8 1196 1197 1198, 1199, 1200, 1201, 1202cyclin_B1 UniProtKB: P14635 1203 1204 1205, 1206, 1207, 1208, 1209cyclin_D1 UniProtKB: P24385 1210 1211 1212, 1213, 1214, 1215, 1216 cyp-BUniProtKB: P23284 1217 1218 1219, 1220, 1221, 1222, 1223 DAM-10UniProtKB: P43366 1224 1225 1226, 1227, 1228, 1229, 1230 DEP1AUniProtKB: Q5TB30 1231 1232 1233, 1234, 1235, 1236, 1237 E7 UniProtKB:P03129 1238 1239 1240, 1241, 1242, 1243, 1244 E7 UniProtKB: P06788 12451246 1247, 1248, 1249, 1250, 1251 E7 UniProtKB: P17387 1252 1253 1254,1255, 1256, 1257, 1258 E7 UniProtKB: P06429 1259 1260 1261, 1262, 1263,1264, 1265 E7 UniProtKB: P27230 1266 1267 1268, 1269, 1270, 1271, 1272E7 UniProtKB: P24837 1273 1274 1275, 1276, 1277, 1278, 1279 E7UniProtKB: P21736 1280 1281 1282, 1283, 1284, 1285, 1286 E7 UniProtKB:P26558 1287 1288 1289, 1290, 1291, 1292, 1293 E7 UniProtKB: P36831 12941295 1296, 1297, 1298, 1299, 1300 E7 UniProtKB: P36833 1301 1302 1303,1304, 1305, 1306, 1307 E7 UniProtKB: Q9QCZ1 1308 1309 1310, 1311, 1312,1313, 1314 E7 UniProtKB: Q81965 1315 1316 1317, 1318, 1319, 1320, 1321E7 UniProtKB: Q80956 1322 1323 1324, 1325, 1326, 1327, 1328 EF1A2UniProtKB: Q05639 1329 1330 1331, 1332, 1333, 1334, 1335 EFTUD2/mUniProtKB: Q15029 1336 1337 1338, 1339, 1340, 1341, 1342 EGFR UniProtKB:A0A0B4J1Y5 1343 1344 1345, 1346, 1347, 1348, 1349 EGFR UniProtKB: E7BSV01350 1351 1352, 1353, 1354, 1355, 1356 EGFR UniProtKB: L0R6G1 1357 13581359, 1360, 1361, 1362, 1363 EGFR UniProtKB: P00533-2 1364 1365 1366,1367, 1368, 1369, 1370 EGFR UniProtKB: P00533 1371 1372 1373, 1374,1375, 1376, 1377 EGFR UniProtKB: Q147T7 1378 1379 1380, 1381, 1382,1383, 1384 EGFR UniProtKB: Q504U8 1385 1386 1387, 1388, 1389, 1390, 1391EGFR UniProtKB: Q8NDU8 1392 1393 1394, 1395, 1396, 1397, 1398 EGLN3UniProtKB: Q9H6Z9 1399 1400 1401, 1402, 1403, 1404, 1405 ELF2/mUniProtKB: B7Z720 1406 1407 1408, 1409, 1410, 1411, 1412 EMMPRINUniProtKB: Q54A51 1413 1414 1415, 1416, 1417, 1418, 1419 EpCamUniProtKB: P16422 1420 1421 1422, 1423, 1424, 1425, 1426 EphA2UniProtKB: P29317 1427 1428 1429, 1430, 1431, 1432, 1433 EphA3UniProtKB: P29320 1434 1435 1436, 1437, 1438, 1439, 1440 EphA3UniProtKB: Q6P4R6 1441 1442 1443, 1444, 1445, 1446, 1447 ErbB3UniProtKB: B3KWG5 1448 1449 1450, 1451, 1452, 1453, 1454 ErbB3UniProtKB: B4DGQ7 1455 1456 1457, 1458, 1459, 1460, 1461 ERBB4UniProtKB: Q15303 1462 1463 1464, 1465, 1466, 1467, 1468 ERG UniProtKB:P11308 1469 1470 1471, 1472, 1473, 1474, 1475 ETV6 UniProtKB: P412121476 1477 1478, 1479, 1480, 1481, 1482 EWS UniProtKB: Q01844 1483 14841485, 1486, 1487, 1488, 1489 EZH2 UniProtKB: F2YMM1 1490 1491 1492,1493, 1494, 1495, 1496 EZH2 UniProtKB: G3XAL2 1497 1498 1499, 1500,1501, 1502, 1503 EZH2 UniProtKB: L0R855 1504 1505 1506, 1507, 1508,1509, 1510 EZH2 UniProtKB: Q15910 1511 1512 1513, 1514, 1515, 1516, 1517EZH2 UniProtKB: S4S3R8 1518 1519 1520, 1521, 1522, 1523, 1524 FABP7UniProtKB: O15540 1525 1526 1527, 1528, 1529, 1530, 1531FCGR3A_Version_1 UniProtKB: P08637 1532 1533 1534, 1535, 1536, 1537,1538 FCGR3A_Version_2 CCDS: CCDS1232.1 1539 1540 1541, 1542, 1543, 1544,1545 FGF5 UniProtKB: P12034 1546 1547 1548, 1549, 1550, 1551, 1552 FGF5UniProtKB: Q60518 1553 1554 1555, 1556, 1557, 1558, 1559 FGFR2UniProtKB: P21802 1560 1561 1562, 1563, 1564, 1565, 1566 fibronectinUniProtKB: A0A024R5I6 1567 1568 1569, 1570, 1571, 1572, 1573 fibronectinUniProtKB: A0A024RB01 1574 1575 1576, 1577, 1578, 1579, 1580 fibronectinUniProtKB: A0A024RDT9 1581 1582 1583, 1584, 1585, 1586, 1587 fibronectinUniProtKB: A0A024RDV5 1588 1589 1590, 1591, 1592, 1593, 1594 fibronectinUniProtKB: A6NH44 1595 1596 1597, 1598, 1599, 1600, 1601 fibronectinUniProtKB: A8K6A5 1602 1603 1604, 1605, 1606, 1607, 1608 fibronectinUniProtKB: B2R627 1609 1610 1611, 1612, 1613, 1614, 1615 fibronectinUniProtKB: B3KXM5 1616 1617 1618, 1619, 1620, 1621, 1622 fibronectinUniProtKB: B4DIC5 1623 1624 1625, 1626, 1627, 1628, 1629 fibronectinUniProtKB: B4DN21 1630 1631 1632, 1633, 1634, 1635, 1636 fibronectinUniProtKB: B4DS98 1637 1638 1639, 1640, 1641, 1642, 1643 fibronectinUniProtKB: B4DTH2 1644 1645 1646, 1647, 1648, 1649, 1650 fibronectinUniProtKB: B4DTK1 1651 1652 1653, 1654, 1655, 1656, 1657 fibronectinUniProtKB: B4DU16 1658 1659 1660, 1661, 1662, 1663, 1664 fibronectinUniProtKB: B7Z3W5 1665 1666 1667, 1668, 1669, 1670, 1671 fibronectinUniProtKB: B7Z939 1672 1673 1674, 1675, 1676, 1677, 1678 fibronectinUniProtKB: G5E9X3 1679 1680 1681, 1682, 1683, 1684, 1685 fibronectinUniProtKB: Q9H382 1686 1687 1688, 1689, 1690, 1691, 1692 FOS UniProtKB:P01100 1693 1694 1695, 1696, 1697, 1698, 1699 FOXP3 UniProtKB: Q9BZS11700 1701 1702, 1703, 1704, 1705, 1706 FUT1 UniProtKB: P19526 1707 17081709, 1710, 1711, 1712, 1713 G250 UniProtKB: Q16790 1714 1715 1716,1717, 1718, 1719, 1720 GAGE-1 Genbank: AAA82744 1721 1722 1723, 1724,1725, 1726, 1727 GAGE-2 UniProtKB: Q6NT46 1728 1729 1730, 1731, 1732,1733, 1734 GAGE-3 UniProtKB: Q13067 1735 1736 1737, 1738, 1739, 1740,1741 GAGE-4 UniProtKB: Q13068 1742 1743 1744, 1745, 1746, 1747, 1748GAGE-5 UniProtKB: Q13069 1749 1750 1751, 1752, 1753, 1754, 1755 GAGE-6UniProtKB: Q13070 1756 1757 1758, 1759, 1760, 1761, 1762 GAGE7bUniProtKB: O76087 1763 1764 1765, 1766, 1767, 1768, 1769GAGE-8_(GAGE-2D) UniProtKB: Q9UEU5 1770 1771 1772, 1773, 1774, 1775,1776 GASR UniProtKB: P32239 1777 1778 1779, 1780, 1781, 1782, 1783 GnT-VUniProtKB: Q09328 1784 1785 1786, 1787, 1788, 1789, 1790 GPC3 UniProtKB:I6QJG3 1791 1792 1793, 1794, 1795, 1796, 1797 GPC3 UniProtKB: P516541798 1799 1800, 1801, 1802, 1803, 1804 GPC3 UniProtKB: Q8IYG2 1805 18061807, 1808, 1809, 1810, 1811 GPNMB/m UniProtKB: A0A024RA55 1812 18131814, 1815, 1816, 1817, 1818 GPNMB/m UniProtKB: Q14956 1819 1820 1821,1822, 1823, 1824, 1825 GPNMB/m UniProtKB: Q8IXJ5 1826 1827 1828, 1829,1830, 1831, 1832 GPNMB/m UniProtKB: Q96F58 1833 1834 1835, 1836, 1837,1838, 1839 GRM3 UniProtKB: Q14832 1840 1841 1842, 1843, 1844, 1845, 1846HAGE UniProtKB: Q9NXZ2 1847 1848 1849, 1850, 1851, 1852, 1853 hepsinUniProtKB: B2ZDQ2 1854 1855 1856, 1857, 1858, 1859, 1860 hepsinUniProtKB: P05981 1861 1862 1863, 1864, 1865, 1866, 1867 Her2/neuUniProtKB: B4DTR1 1868 1869 1870, 1871, 1872, 1873, 1874 Her2/neuUniProtKB: L8E8G2 1875 1876 1877, 1878, 1879, 1880, 1881 Her2/neuUniProtKB: P04626 1882 1883 1884, 1885, 1886, 1887, 1888 Her2/neuUniProtKB: Q9UK79 1889 1890 1891, 1892, 1893, 1894, 1895 HLA-A2/mUniProtKB: Q95387 1896 1897 1898, 1899, 1900, 1901, 1902 HLA-A2/mUniProtKB: Q9MYF8 1903 1904 1905, 1906, 1907, 1908, 1909 homeobox_NKX3.1UniProtKB: Q99801 1910 1911 1912, 1913, 1914, 1915, 1916 HOM-TES-85UniProtKB: B2RBQ6 1917 1918 1919, 1920, 1921, 1922, 1923 HOM-TES-85UniProtKB: Q9P127 1924 1925 1926, 1927, 1928, 1929, 1930 HPG1 Pubmed:12543784 1931 1932 1933, 1934, 1935, 1936, 1937 HS71A UniProtKB: P0DMV81938 1939 1940, 1941, 1942, 1943, 1944 HS71B UniProtKB: P0DMV9 1945 19461947, 1948, 1949, 1950, 1951 HST-2 UniProtKB: P10767 1952 1953 1954,1955, 1956, 1957, 1958 hTERT UniProtKB: O94807 1959 1960 1961, 1962,1963, 1964, 1965 iCE UniProtKB: O00748 1966 1967 1968, 1969, 1970, 1971,1972 IF2B3 UniProtKB: O00425 1973 1974 1975, 1976, 1977, 1978, 1979IL-13Ra2 UniProtKB: Q14627 1980 1981 1982, 1983, 1984, 1985, 1986 IL2-RAUniProtKB: P01589 1987 1988 1989, 1990, 1991, 1992, 1993 IL2-RBUniProtKB: P14784 1994 1995 1996, 1997, 1998, 1999, 2000 IL2-RGUniProtKB: P31785 2001 2002 2003, 2004, 2005, 2006, 2007 IMP3 UniProtKB:Q9NV31 2008 2009 2010, 2011, 2012, 2013, 2014 ITA5 UniProtKB: P086482015 2016 2017, 2018, 2019, 2020, 2021 ITB1 UniProtKB: P05556 2022 20232024, 2025, 2026, 2027, 2028 ITB6 UniProtKB: P18564 2029 2030 2031,2032, 2033, 2034, 2035 kallikrein-2 UniProtKB: A0A024R4J4 2036 20372038, 2039, 2040, 2041, 2042 kallikrein-2 UniProtKB: A0A024R4N3 20432044 2045, 2046, 2047, 2048, 2049 kallikrein-2 UniProtKB: B0AZU9 20502051 2052, 2053, 2054, 2055, 2056 kallikrein-2 UniProtKB: B4DU77 20572058 2059, 2060, 2061, 2062, 2063 kallikrein-2 UniProtKB: P20151 20642065 2066, 2067, 2068, 2069, 2070 kallikrein-2 UniProtKB: Q6T774 20712072 2073, 2074, 2075, 2076, 2077 kallikrein-2 UniProtKB: Q6T775 20782079 2080, 2081, 2082, 2083, 2084 kallikrein-4 UniProtKB: A0A0C4DFQ52085 2086 2087, 2088, 2089, 2090, 2091 kallikrein-4 UniProtKB: Q5BQA02092 2093 2094, 2095, 2096, 2097, 2098 kallikrein-4 UniProtKB: Q96PT02099 2100 2101, 2102, 2103, 2104, 2105 kallikrein-4 UniProtKB: Q96PT12106 2107 2108, 2109, 2110, 2111, 2112 kallikrein-4 UniProtKB: Q9Y5K22113 2114 2115, 2116, 2117, 2118, 2119 KI20A UniProtKB: O95235 2120 21212122, 2123, 2124, 2125, 2126 KIAA0205 UniProtKB: Q92604 2127 2128 2129,2130, 2131, 2132, 2133 KIF2C UniProtKB: Q99661 2134 2135 2136, 2137,2138, 2139, 2140 KK-LC-1 UniProtKB: Q5H943 2141 2142 2143, 2144, 2145,2146, 2147 LDLR UniProtKB: P01130 2148 2149 2150, 2151, 2152, 2153, 2154LGMN UniProtKB: Q99538 2155 2156 2157, 2158, 2159, 2160, 2161 LIRB2UniProtKB: Q8N423 2162 2163 2164, 2165, 2166, 2167, 2168 LY6K UniProtKB:Q17RY6 2169 2170 2171, 2172, 2173, 2174, 2175 MAGA5 UniProtKB: P433592176 2177 2178, 2179, 2180, 2181, 2182 MAGA8 UniProtKB: P43361 2183 21842185, 2186, 2187, 2188, 2189 MAGAB UniProtKB: P43364 2190 2191 2192,2193, 2194, 2195, 2196 MAGE-A10 UniProtKB: A0A024RC14 2197 2198 2199,2200, 2201, 2202, 2203 MAGE-A12 UniProtKB: P43365 2204 2205 2206, 2207,2208, 2209, 2210 MAGE-A1 UniProtKB: P43355 2211 2212 2213, 2214, 2215,2216, 2217 MAGE-A2 UniProtKB: P43356 2218 2219 2220, 2221, 2222, 2223,2224 MAGE-A3 UniProtKB: P43357 2225 2226 2227, 2228, 2229, 2230, 2231MAGE-A4 UniProtKB: A0A024RC12 2232 2233 2234, 2235, 2236, 2237, 2238MAGE-A4 UniProtKB: P43358 2239 2240 2241, 2242, 2243, 2244, 2245 MAGE-A4UniProtKB: Q1RN33 2246 2247 2248, 2249, 2250, 2251, 2252 MAGE-A6UniProtKB: A8K072 2253 2254 2255, 2256, 2257, 2258, 2259 MAGE-A6UniProtKB: P43360 2260 2261 2262, 2263, 2264, 2265, 2266 MAGE-A6UniProtKB: Q6FHI5 2267 2268 2269, 2270, 2271, 2272, 2273 MAGE-A9UniProtKB: P43362 2274 2275 2276, 2277, 2278, 2279, 2280 MAGE-B10UniProtKB: Q96LZ2 2281 2282 2283, 2284, 2285, 2286, 2287 MAGE-B16UniProtKB: A2A368 2288 2289 2290, 2291, 2292, 2293, 2294 MAGE-B17UniProtKB: A8MXT2 2295 2296 2297, 2298, 2299, 2300, 2301 MAGE-_B1UniProtKB: Q96TG1 2302 2303 2304, 2305, 2306, 2307, 2308 MAGE-B2UniProtKB: O15479 2309 2310 2311, 2312, 2313, 2314, 2315 MAGE-B3UniProtKB: O15480 2316 2317 2318, 2319, 2320, 2321, 2322 MAGE-B4UniProtKB: O15481 2323 2324 2325, 2326, 2327, 2328, 2329 MAGE-B5UniProtKB: Q9BZ81 2330 2331 2332, 2333, 2334, 2335, 2336 MAGE-B6UniProtKB: Q8N7X4 2337 2338 2339, 2340, 2341, 2342, 2343 MAGE-C1UniProtKB: O60732 2344 2345 2346, 2347, 2348, 2349, 2350 MAGE-C2UniProtKB: Q9UBF1 2351 2352 2353, 2354, 2355, 2356, 2357 MAGE-C3UniProtKB: Q8TD91 2358 2359 2360, 2361, 2362, 2363, 2364 MAGE-D1UniProtKB: Q9Y5V3 2365 2366 2367, 2368, 2369, 2370, 2371 MAGE-D2UniProtKB: Q9UNF1 2372 2373 2374, 2375, 2376, 2377, 2378 MAGE-D4UniProtKB: Q96JG8 2379 2380 2381, 2382, 2383, 2384, 2385 MAGE-_E1UniProtKB: Q6IAI7 2386 2387 2388, 2389, 2390, 2391, 2392 MAGE-E1_(MAGE1)UniProtKB: Q9HCI5 2393 2394 2395, 2396, 2397, 2398, 2399 MAGE-E2UniProtKB: Q8TD90 2400 2401 2402, 2403, 2404, 2405, 2406 MAGE-F1UniProtKB: Q9HAY2 2407 2408 2409, 2410, 2411, 2412, 2413 MAGE-H1UniProtKB: Q9H213 2414 2415 2416, 2417, 2418, 2419, 2420 MAGEL2UniProtKB: Q9UJ55 2421 2422 2423, 2424, 2425, 2426, 2427 mammaglobin_AUniProtKB: Q13296 2428 2429 2430, 2431, 2432, 2433, 2434 mammaglobin_AUniProtKB: Q6NX70 2435 2436 2437, 2438, 2439, 2440, 2441 MART-1/melan-AUniProtKB: Q16655 2442 2443 2444, 2445, 2446, 2447, 2448 MART-2UniProtKB: Q5VTY9 2449 2450 2451, 2452, 2453, 2454, 2455 MC1_RUniProtKB: Q01726 2456 2457 2458, 2459, 2460, 2461, 2462 MC1_RUniProtKB: Q1JUL4 2463 2464 2465, 2466, 2467, 2468, 2469 MC1_RUniProtKB: Q1JUL6 2470 2471 2472, 2473, 2474, 2475, 2476 MC1_RUniProtKB: Q1JUL8 2477 2478 2479, 2480, 2481, 2482, 2483 MC1_RUniProtKB: Q1JUL9 2484 2485 2486, 2487, 2488, 2489, 2490 MC1_RUniProtKB: Q1JUM0 2491 2492 2493, 2494, 2495, 2496, 2497 MC1_RUniProtKB: Q1JUM2 2498 2499 2500, 2501, 2502, 2503, 2504 MC1_RUniProtKB: Q1JUM3 2505 2506 2507, 2508, 2509, 2510, 2511 MC1_RUniProtKB: Q1JUM4 2512 2513 2514, 2515, 2516, 2517, 2518 MC1_RUniProtKB: Q1JUM5 2519 2520 2521, 2522, 2523, 2524, 2525 MC1_RUniProtKB: Q6UR92 2526 2527 2528, 2529, 2530, 2531, 2532 MC1_RUniProtKB: Q6UR94 2533 2534 2535, 2536, 2537, 2538, 2539 MC1_RUniProtKB: Q6UR95 2540 2541 2542, 2543, 2544, 2545, 2546 MC1_RUniProtKB: Q6UR96 2547 2548 2549, 2550, 2551, 2552, 2553 MC1_RUniProtKB: Q6UR97 2554 2555 2556, 2557, 2558, 2559, 2560 MC1_RUniProtKB: Q6UR98 2561 2562 2563, 2564, 2565, 2566, 2567 MC1_RUniProtKB: Q6UR99 2568 2569 2570, 2571, 2572, 2573, 2574 MC1_RUniProtKB: Q6URA0 2575 2576 2577, 2578, 2579, 2580, 2581 MC1_RUniProtKB: Q86YW1 2582 2583 2584, 2585, 2586, 2587, 2588 MC1_RUniProtKB: V9Q5S2 2589 2590 2591, 2592, 2593, 2594, 2595 MC1_RUniProtKB: V9Q671 2596 2597 2598, 2599, 2600, 2601, 2602 MC1_RUniProtKB: V9Q783 2603 2604 2605, 2606, 2607, 2608, 2609 MC1_RUniProtKB: V9Q7F1 2610 2611 2612, 2613, 2614, 2615, 2616 MC1_RUniProtKB: V9Q8N1 2617 2618 2619, 2620, 2621, 2622, 2623 MC1_RUniProtKB: V9Q977 2624 2625 2626, 2627, 2628, 2629, 2630 MC1_RUniProtKB: V9Q9P5 2631 2632 2633, 2634, 2635, 2636, 2637 MC1_RUniProtKB: V9Q9R8 2638 2639 2640, 2641, 2642, 2643, 2644 MC1_RUniProtKB: V9QAE0 2645 2646 2647, 2648, 2649, 2650, 2651 MC1_RUniProtKB: V9QAR2 2652 2653 2654, 2655, 2656, 2657, 2658 MC1_RUniProtKB: V9QAW3 2659 2660 2661, 2662, 2663, 2664, 2665 MC1_RUniProtKB: V9QB02 2666 2667 2668, 2669, 2670, 2671, 2672 MC1_RUniProtKB: V9QB58 2673 2674 2675, 2676, 2677, 2678, 2679 MC1_RUniProtKB: V9QBY6 2680 2681 2682, 2683, 2684, 2685, 2686 MC1_RUniProtKB: V9QC17 2687 2688 2689, 2690, 2691, 2692, 2693 MC1_RUniProtKB: V9QC66 2694 2695 2696, 2697, 2698, 2699, 2700 MC1_RUniProtKB: V9QCQ4 2701 2702 2703, 2704, 2705, 2706, 2707 MC1_RUniProtKB: V9QDF4 2708 2709 2710, 2711, 2712, 2713, 2714 MC1_RUniProtKB: V9QDN7 2715 2716 2717, 2718, 2719, 2720, 2721 MC1_RUniProtKB: V9QDQ6 2722 2723 2724, 2725, 2726, 2727, 2728 mesothelinUniProtKB: Q13421 2729 2730 2731, 2732, 2733, 2734, 2735 MITF UniProtKB:O75030-8 2736 2737 2738, 2739, 2740, 2741, 2742 MITF UniProtKB: O75030-92743 2744 2745, 2746, 2747, 2748, 2749 MITF UniProtKB: O75030 2750 27512752, 2753, 2754, 2755, 2756 MMP1_1 UniProtKB: B3KQS8 2757 2758 2759,2760, 2761, 2762, 2763 MMP7 UniProtKB: P09237 2764 2765 2766, 2767,2768, 2769, 2770 MUC-1 Genbank: AAA60019 2771 2772 2773, 2774, 2775,2776, 2777 MUM-1/m RefSeq: NP_116242 2778 2779 2780, 2781, 2782, 2783,2784 MUM-2/m UniProtKB: Q9Y5R8 2785 2786 2787, 2788, 2789, 2790, 2791MYO1A UniProtKB: Q9UBC5 2792 2793 2794, 2795, 2796, 2797, 2798 MYO1BUniProtKB: O43795 2799 2800 2801, 2802, 2803, 2804, 2805 MYO1CUniProtKB: O00159 2806 2807 2808, 2809, 2810, 2811, 2812 MYO1DUniProtKB: O94832 2813 2814 2815, 2816, 2817, 2818, 2819 MYO1EUniProtKB: Q12965 2820 2821 2822, 2823, 2824, 2825, 2826 MYO1FUniProtKB: O00160 2827 2828 2829, 2830, 2831, 2832, 2833 MYO1GUniProtKB: B0I1T2 2834 2835 2836, 2837, 2838, 2839, 2840 MYO1H RefSeq:NP_001094891 2841 2842 2843, 2844, 2845, 2846, 2847 NA17 UniProtKB:Q3V5L5 2848 2849 2850, 2851, 2852, 2853, 2854 NA88-A Pubmed: 107904362855 2856 2857, 2858, 2859, 2860, 2861 Neo-PAP UniProtKB: Q9BWT3 28622863 2864, 2865, 2866, 2867, 2868 NFYC/m UniProtKB: Q13952 2869 28702871, 2872, 2873, 2874, 2875 NGEP UniProtKB: Q6IWH7 2876 2877 2878,2879, 2880, 2881, 2882 NPM UniProtKB: P06748 2883 2884 2885, 2886, 2887,2888, 2889 NRCAM UniProtKB: Q92823 2890 2891 2892, 2893, 2894, 2895,2896 NSE UniProtKB: P09104 2897 2898 2899, 2900, 2901, 2902, 2903 NUF2UniProtKB: Q9BZD4 2904 2905 2906, 2907, 2908, 2909, 2910 NY-ESO-1UniProtKB: P78358 2911 2912 2913, 2914, 2915, 2916, 2917 OA1 UniProtKB:P51810 2918 2919 2920, 2921, 2922, 2923, 2924 OGT UniProtKB: O15294 29252926 2927, 2928, 2929, 2930, 2931 OS-9 UniProtKB: B4DH11 2932 2933 2934,2935, 2936, 2937, 2938 OS-9 UniProtKB: B4E321 2939 2940 2941, 2942,2943, 2944, 2945 OS-9 UniProtKB: B7Z8E7 2946 2947 2948, 2949, 2950,2951, 2952 OS-9 UniProtKB: Q13438 2953 2954 2955, 2956, 2957, 2958, 2959osteocalcin UniProtKB: P02818 2960 2961 2962, 2963, 2964, 2965, 2966osteopontin UniProtKB: A0A024RDE2 2967 2968 2969, 2970, 2971, 2972, 2973osteopontin UniProtKB: A0A024RDE6 2974 2975 2976, 2977, 2978, 2979, 2980osteopontin UniProtKB: A0A024RDJ0 2981 2982 2983, 2984, 2985, 2986, 2987osteopontin UniProtKB: B7Z351 2988 2989 2990, 2991, 2992, 2993, 2994osteopontin UniProtKB: F2YQ21 2995 2996 2997, 2998, 2999, 3000, 3001osteopontin UniProtKB: P10451 3002 3003 3004, 3005, 3006, 3007, 3008 p53UniProtKB: P04637 3009 3010 3011, 3012, 3013, 3014, 3015 PAGE-4UniProtKB: O60829 3016 3017 3018, 3019, 3020, 3021, 3022 PAI-1UniProtKB: P05121 3023 3024 3025, 3026, 3027, 3028, 3029 PAI-2UniProtKB: P05120 3030 3031 3032, 3033, 3034, 3035, 3036 PAP UniProtKB:Q06141 3037 3038 3039, 3040, 3041, 3042, 3043 PAP UniProtKB: Q53S56 30443045 3046, 3047, 3048, 3049, 3050 PATE UniProtKB: Q8WXA2 3051 3052 3053,3054, 3055, 3056, 3057 PAX3 UniProtKB: P23760 3058 3059 3060, 3061,3062, 3063, 3064 PAX5 UniProtKB: Q02548 3065 3066 3067, 3068, 3069,3070, 3071 PD1L1 UniProtKB: Q9NZQ7 3072 3073 3074, 3075, 3076, 3077,3078 PDCD1 UniProtKB: Q15116 3079 3080 3081, 3082, 3083, 3084, 3085 PDEFUniProtKB: O95238 3086 3087 3088, 3089, 3090, 3091, 3092 PECA1UniProtKB: P16284 3093 3094 3095, 3096, 3097, 3098, 3099 PGCB UniProtKB:Q96GW7 3100 3101 3102, 3103, 3104, 3105, 3106 PGFRB UniProtKB: P096193107 3108 3109, 3110, 3111, 3112, 3113 Pim-1_-Kinase UniProtKB:A0A024RD25 3114 3115 3116, 3117, 3118, 3119, 3120 Pin-1 UniProtKB:O15428 3121 3122 3123, 3124, 3125, 3126, 3127 Pin-1 UniProtKB: Q135263128 3129 3130, 3131, 3132, 3133, 3134 Pin-1 UniProtKB: Q49AR7 3135 31363137, 3138, 3139, 3140, 3141 PLAC1 UniProtKB: Q9HBJ0 3142 3143 3144,3145, 3146, 3147, 3148 PMEL UniProtKB: P40967 3149 3150 3151, 3152,3153, 3154, 3155 PML UniProtKB: P29590 3156 3157 3158, 3159, 3160, 3161,3162 POTEF UniProtKB: A5A3E0 3163 3164 3165, 3166, 3167, 3168, 3169 POTEUniProtKB: Q86YR6 3170 3171 3172, 3173, 3174, 3175, 3176 PRAMEUniProtKB: A0A024R1E6 3177 3178 3179, 3180, 3181, 3182, 3183 PRAMEUniProtKB: P78395 3184 3185 3186, 3187, 3188, 3189, 3190 PRDX5/mUniProtKB: P30044 3191 3192 3193, 3194, 3195, 3196, 3197 PRM2 UniProtKB:P04554 3198 3199 3200, 3201, 3202, 3203, 3204 prostein UniProtKB: Q96JT23205 3206 3207, 3208, 3209, 3210, 3211 proteinase-3 UniProtKB: D6CHE93212 3213 3214, 3215, 3216, 3217, 3218 proteinase-3 UniProtKB: P241583219 3220 3221, 3222, 3223, 3224, 3225 PSA UniProtKB: P55786 3226 32273228, 3229, 3230, 3231, 3232 PSB9 UniProtKB: P28065 3233 3234 3235,3236, 3237, 3238, 3239 PSCA UniProtKB: D3DWI6 3240 3241 3242, 3243,3244, 3245, 3246 PSCA UniProtKB: O43653 3247 3248 3249, 3250, 3251,3252, 3253 PSGR UniProtKB: Q9H255 3254 3255 3256, 3257, 3258, 3259, 3260PSM UniProtKB: Q04609 3261 3262 3263, 3264, 3265, 3266, 3267 PTPRCRefSeq: NP_002829 3268 3269 3270, 3271, 3272, 3273, 3274 RAB8AUniProtKB: P61006 3275 3276 3277, 3278, 3279, 3280, 3281 RAGE-1UniProtKB: Q9UQ07 3282 3283 3284, 3285, 3286, 3287, 3288 RARA UniProtKB:P10276 3289 3290 3291, 3292, 3293, 3294, 3295 RASH UniProtKB: P011123296 3297 3298, 3299, 3300, 3301, 3302 RASK UniProtKB: P01116 3303 33043305, 3306, 3307, 3308, 3309 RASN UniProtKB: P01111 3310 3311 3312,3313, 3314, 3315, 3316 RGS5 UniProtKB: O15539 3317 3318 3319, 3320,3321, 3322, 3323 RHAMM/CD168 UniProtKB: O75330 3324 3325 3326, 3327,3328, 3329, 3330 RHOC UniProtKB: P08134 3331 3332 3333, 3334, 3335,3336, 3337 RSSA UniProtKB: P08865 3338 3339 3340, 3341, 3342, 3343, 3344RU1 UniProtKB: Q9UHJ3 3345 3346 3347, 3348, 3349, 3350, 3351 RU2UniProtKB: Q9UHG0 3352 3353 3354, 3355, 3356, 3357, 3358 RUNX1UniProtKB: Q01196 3359 3360 3361, 3362, 3363, 3364, 3365 S-100UniProtKB: V9HW39 3366 3367 3368, 3369, 3370, 3371, 3372 SAGE UniProtKB:Q9NXZ1 3373 3374 3375, 3376, 3377, 3378, 3379 SART-_1 UniProtKB: O432903380 3381 3382, 3383, 3384, 3385, 3386 SART-2 UniProtKB: Q9UL01 33873388 3389, 3390, 3391, 3392, 3393 SART-3 UniProtKB: Q15020 3394 33953396, 3397, 3398, 3399, 3400 SEPR UniProtKB: Q12884 3401 3402 3403,3404, 3405, 3406, 3407 SIA7F UniProtKB: Q969X2 3408 3409 3410, 3411,3412, 3413, 3414 SIA8A UniProtKB: Q92185 3415 3416 3417, 3418, 3419,3420, 3421 SIAT9 UniProtKB: Q9UNP4 3422 3423 3424, 3425, 3426, 3427,3428 SIRT2/m UniProtKB: A0A024R0G8 3429 3430 3431, 3432, 3433, 3434,3435 SIRT2/m UniProtKB: Q8IXJ6 3436 3437 3438, 3439, 3440, 3441, 3442SOX10 UniProtKB: P56693 3443 3444 3445, 3446, 3447, 3448, 3449 SP17UniProtKB: Q15506 3450 3451 3452, 3453, 3454, 3455, 3456 SPNXAUniProtKB: Q9NS26 3457 3458 3459, 3460, 3461, 3462, 3463 SPXN3UniProtKB: Q5MJ09 3464 3465 3466, 3467, 3468, 3469, 3470 SSX-1UniProtKB: Q16384 3471 3472 3473, 3474, 3475, 3476, 3477 SSX-2UniProtKB: Q16385 3478 3479 3480, 3481, 3482, 3483, 3484 SSX3 UniProtKB:Q99909 3485 3486 3487, 3488, 3489, 3490, 3491 SSX-4 UniProtKB: O602243492 3493 3494, 3495, 3496, 3497, 3498 ST1A1 UniProtKB: P50225 3499 35003501, 3502, 3503, 3504, 3505 STAG2 UniProtKB: Q8N3U4-2 3506 3507 3508,3509, 3510, 3511, 3512 STAMP-1 UniProtKB: Q8NFT2 3513 3514 3515, 3516,3517, 3518, 3519 STEAP-1 UniProtKB: A0A024RA63 3520 3521 3522, 3523,3524, 3525, 3526 STEAP-1 UniProtKB: Q9UHE8 3527 3528 3529, 3530, 3531,3532, 3533 Survivin-2B UniProtKB: O15392-2 3534 3535 3536, 3537, 3538,3539, 3540 survivin UniProtKB: O15392 3541 3542 3543, 3544, 3545, 3546,3547 SYCP1 UniProtKB: A0A024R0I2 3548 3549 3550, 3551, 3552, 3553, 3554SYCP1 UniProtKB: B7ZLS9 3555 3556 3557, 3558, 3559, 3560, 3561 SYCP1UniProtKB: Q15431 3562 3563 3564, 3565, 3566, 3567, 3568 SYCP1UniProtKB: Q3MHC4 3569 3570 3571, 3572, 3573, 3574, 3575 SYT-SSX-1UniProtKB: A4PIV7 3576 3577 3578, 3579, 3580, 3581, 3582 SYT-SSX-1UniProtKB: A4PIV8 3583 3584 3585, 3586, 3587, 3588, 3589 SYT-SSX-2UniProtKB: A4PIV9 3590 3591 3592, 3593, 3594, 3595, 3596 SYT-SSX-2UniProtKB: A4PIW0 3597 3598 3599, 3600, 3601, 3602, 3603 TARP UniProtKB:Q0VGM3 3604 3605 3606, 3607, 3608, 3609, 3610 TCRg UniProtKB: A2JGV33611 3612 3613, 3614, 3615, 3616, 3617 TF2AA UniProtKB: P52655 3618 36193620, 3621, 3622, 3623, 3624 TGFR2 UniProtKB: P37173 3625 3626 3627,3628, 3629, 3630, 3631 TGM-4 UniProtKB: B2R7D1 3632 3633 3634, 3635,3636, 3637, 3638 TIE2 UniProtKB: Q02763 3639 3640 3641, 3642, 3643,3644, 3645 TKTL1 UniProtKB: P51854 3646 3647 3648, 3649, 3650, 3651,3652 TPI/m UniProtKB: P60174 3653 3654 3655, 3656, 3657, 3658, 3659TRGV11 UniProtKB: Q99601 3660 3661 3662, 3663, 3664, 3665, 3666 TRGV9UniProtKB: A4D1X2 3667 3668 3669, 3670, 3671, 3672, 3673 TRGV9UniProtKB: Q99603 3674 3675 3676, 3677, 3678, 3679, 3680 TRGV9UniProtKB: Q99604 3681 3682 3683, 3684, 3685, 3686, 3687 TRPC1UniProtKB: P48995 3688 3689 3690, 3691, 3692, 3693, 3694 TRP-p8UniProtKB: Q7Z2W7 3695 3696 3697, 3698, 3699, 3700, 3701 TSG10UniProtKB: Q9BZW7 3702 3703 3704, 3705, 3706, 3707, 3708 TSPY1UniProtKB: Q01534 3709 3710 3711, 3712, 3713, 3714, 3715 TVC_(TRGV3)Genbank: M13231.1 3716 3717 3718, 3719, 3720, 3721, 3722 TX101UniProtKB: Q9BY14-2 3723 3724 3725, 3726, 3727, 3728, 3729 tyrosinaseUniProtKB: A0A024DBG7 3730 3731 3732, 3733, 3734, 3735, 3736 tyrosinaseUniProtKB: L8B082 3737 3738 3739, 3740, 3741, 3742, 3743 tyrosinaseUniProtKB: L8B086 3744 3745 3746, 3747, 3748, 3749, 3750 tyrosinaseUniProtKB: L8B0B9 3751 3752 3753, 3754, 3755, 3756, 3757 tyrosinaseUniProtKB: O75767 3758 3759 3760, 3761, 3762, 3763, 3764 tyrosinaseUniProtKB: P14679 3765 3766 3767, 3768, 3769, 3770, 3771 tyrosinaseUniProtKB: U3M8N0 3772 3773 3774, 3775, 3776, 3777, 3778 tyrosinaseUniProtKB: U3M9D5 3779 3780 3781, 3782, 3783, 3784, 3785 tyrosinaseUniProtKB: U3M9J2 3786 3787 3788, 3789, 3790, 3791, 3792 TYRP1UniProtKB: P17643 3793 3794 3795, 3796, 3797, 3798, 3799 TYRP2UniProtKB: P40126 3800 3801 3802, 3803, 3804, 3805, 3806 UPA UniProtKB:Q96NZ9 3807 3808 3809, 3810, 3811, 3812, 3813 VEGFR1 UniProtKB: B5A9243814 3815 3816, 3817, 3818, 3819, 3820 WT1 UniProtKB: A0A0H5AUY0 38213822 3823, 3824, 3825, 3826, 3827 WT1 UniProtKB: P19544 3828 3829 3830,3831, 3832, 3833, 3834 WT1 UniProtKB: Q06250 3835 3836 3837, 3838, 3839,3840, 3841 XAGE1 UniProtKB: Q9HD64 3842 3843 3844, 3845, 3846, 3847,3848 IL-10 UniProtKB: P22301 4169 4170 4171, 4172, 4173, 4174, 4175,4176 IL-5 UniProtKB: P05113 4585 4586 4587, 4588, 4589, 4590, 4591, 4592M-CSF UniProtKB: P09603 4705 4706 4707, 4708, 4709, 4710, 4711, 4712TGFbeta1 UniProtKB: P01137 4785 4786 4787, 4788, 4789, 4790, 4791, 4792Caspase_8 UniProtKB: Q14790 7113 7114 7115, 7116, 7117, 7118, 7119, 7120SERPINB5 UniProtKB: P36952 7465 7466 7467, 7468, 7469, 7470, 7471, 7472calreticulin UniProtKB: B4DHR1 7569 7570 7571, 7572, 7573, 7574, 7575,7576 calreticulin UniProtKB: B4E2Y9 7577 7578 7579, 7580, 7581, 7582,7583, 7584 calreticulin UniProtKB: P27797 7585 7586 7587, 7588, 7589,7590, 7591, 7592 calreticulin UniProtKB: Q96L12 7593 7594 7595, 7596,7597, 7598, 7599, 7600 N-myc UniProtKB: P04198 9987 9988 9989, 9990,9991, 9992, 9993, 9994

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one tumorantigen or a fragment or variant thereof, wherein the at least onecoding region comprises an RNA sequence being identical or at least 50%,60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNAsequences according to the SEQ ID Nos as disclosed in Table 9.

Furthermore tumor antigens also may encompass idiotypic antigensassociated with a cancer or tumor disease, particularly lymphoma or alymphoma associated disease, wherein said idiotypic antigen is animmunoglobulin idiotype of a lymphoid blood cell or a T cell receptoridiotype of a lymphoid blood cell.

In a particularly preferred embodiment the inventive RNA compositioncomprises at least one RNA, wherein the at least one RNA encodes thefollowing antigens:

-   -   STEAP (Six Transmembrane Epithelial Antigen of the Prostate);    -   PSA (Prostate-Specific Antigen),    -   PSMA (Prostate-Specific Membrane Antigen),    -   PSCA (Prostate Stem Cell Antigen);    -   PAP (Prostatic Acid Phosphatase), and    -   MUC1 (Mucin 1).

In another particularly preferred embodiment the inventive RNAcomposition comprises at least one RNA, wherein the at least one RNAencodes the following antigens:

-   -   5T4 (Trophoblast glycoprotein, TPBG);    -   Survivin (Baculoviral IAP repeat-containing protein 5; BIRC5),    -   NY-ESO-1 (New York esophageal squamous cell carcinoma 1;        CTAG1B),    -   MAGE-C1 (Melanoma antigen family C1);    -   MAGE-C2 (Melanoma antigen family C2), and    -   MUC1 (Mucin 1).

9. β-Catenin Inhibitors

In a further preferred embodiment of the inventive RNA containingcomposition the RNA, preferably mRNA codes for at least one β-catenininhibitor or a fragment or variant thereof. Preferably the RNA encodingthe at least one β-catenin inhibitor encodes an inhibitory protein ordominant negative mutant protein of the β-catenin pathway. Particularpreferred β-catenin inhibitors according to the present inventioncomprise TAT-NLS-BLBD-6, axin-1, TCF-4, GSK-3b, DKK-1, Dvl-1 derivativesor fragments thereof.

As reviewed by Thakur and Mishra (Thakur R, Mishra D P. Pharmacologicalmodulation of beta-catenin and its applications in cancer therapy. JCell Mol Med. 2013 April; 17(4):449-56. doi: 10.1111/jcmm.12033)beta-catenin (β-catenin) is a multifunctional protein which plays animportant role in physiological homeostasis. It acts both as atranscriptional regulator and an adaptor protein for intracellularadhesion. β-catenin is necessary for the establishment and maintenanceof epithelial layers and provides a linkage between intracellularjunctions and cytoskeletal proteins. β-catenin is regulated by Wntsignaling. In the absence of Wnt downstream signal β-catenin isphosphorylated which leads to its ubiquitination and eventually proteindegradation. Various literature reports have linked β-catenin to themalignant transformation of normal cells. For example, Wnt signaling andβ-catenin nuclear localization was associated with differentiation ofhepatocytes into a tumoral phenotype. Similarly, in lung epithelial andpancreatic cells, activation of β-catenin was sufficient for inductionof oncogenic transformation. In addition to being a driving force ofmalignant transformation, abnormal β-catenin expression and localizationhas been associated with increased metastatic potential. Recently, ithas been shown that β-catenin signaling prevents T cell infiltration andanti-tumor immunity strongly limiting the potential effects ofimmunotherapies. Since β-catenin plays an important and detrimental rolein tumorigenesis, it has been proposed as a putative drug target.

10. STING-Pathway Activators

In a further preferred embodiment of the inventive RNA containingcomposition the RNA, preferably mRNA codes for at least one activator ofthe STING (stimulator of interferon genes) pathway or a fragment orvariant thereof. Preferably, the RNA encoding the at least one activator(stimulator) of the STING pathway encodes an activating protein or aconstitutively active protein of the STING pathway, preferably DDX41,STING, cGAS, IRF3, TBK1 or STAT6 or a fragment or variant thereof.

As reviewed by Woo et al. (Woo S R, Corrales L, Gajewski T F. The STINGpathway and the T cell-inflamed tumor microenvironment. Trends Immunol.2015 Mar. 7. pii: S1471-4906(15)00019-8. doi: 10.1016/j.it.2015.02.003)and Dubensky et al. (Dubensky T W Jr, Kanne D B, Leong M L. Rationale,progress and development of vaccines utilizing STING-activating cyclicdinucleotide adjuvants. Ther Adv Vaccines. 2013 November; 1(4):131-43.doi: 10.1177/2051013613501988) the so-called STING pathway(STING—stimulator of interferon genes) is responsible for sensing ofcytoplasmic DNA and induction of proinflammatory mediators. Afterbinding of DNA in cytoplasm, STING activates signaling via TANK-bindingkinase 1 (TBK-1)/IRF-3 axis which results in production of IFN-β. Thispathway was shown to play an important role in sensing of DNA viruses aswell as some autoimmune disorders. Recent data have identified STINGpathway as absolutely necessary to induce spontaneous T cell primingagainst tumor antigens in vivo. Tumor DNA was detected withintumor-infiltrating DCs, which led to IFN-β production and T cellactivation. Thus, intratumoral application of small molecules STINGpathway agonists has demonstrated their efficacy in tumor-bearinganimals. Agonists of the STING pathway has been also evaluated asvaccine adjuvants showing potency to induce cellular and humoralimmunity in vaccinated hosts.

11. Checkpoint Modulators

In a further preferred embodiment of the inventive RNA containingcomposition the RNA, preferably mRNA comprises at least one codingregion that codes for at least one checkpoint modulator or a fragment orvariant thereof.

Negative regulatory T cell surface molecules were discovered which areupregulated in activated T cells to dampen their activity, resulting inless effective killing of tumor cells. These inhibitory molecules weretermed negative co-stimulatory molecules due to their homology to the Tcell co-stimulatory molecule CD28. These proteins, also referred to asimmune checkpoint proteins, function in multiple pathways including theattenuation of early activation signals, competition for positiveco-stimulation and direct inhibition of antigen presenting cells(Bour-Jordan et al., 2011. Immunol Rev. 241(1):180-205).

In preferred embodiments of the present invention the checkpointmodulator is a modulator of B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2,B7-H3, B7-H4, B7-H6, B7-H7/HHLA2, BTLA, CD28, CD28H/IGPR-1, CTLA-4,ICOS, PD-1, PD-L2/B7-DC, PDCD6, VISTA/B7-H5/PD-1H, BTN1A1/Butyrophilin,BTN2A1, BTN2A2/Butyrophilin 2A2, BTN3A1/2, BTN3A2, BTN3A3,BTNL2/Butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, BTNL10,CD277/BTN3A1, LAIR1, LAIR2, CD96, CD155/PVR, CRTAM, DNAM-1/CD226,Nectin-2/CD112, Nectin-3, TIGIT, LILRA3/CD85e, LILRA4/CD85g/ILT7,LILRB1/CD85j/ILT2, LILRB2/CD85d/ILT4, LILRB3/CD85a/ILT5,LILRB4/CD85k/ILT3, 4-1BB/TNFRSF9/CD137, 4-1BB Ligand/TNFSF9,BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7,CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40 Ligand/TNFSF5,DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14,LIGHT/TNFSF14, Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha,TNF RII/TNFRSF1B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9,CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7,NTB-A/SLAMF6, SLAM/CD150, TIM-1/KIM-1/HAVCR, TIM-3, TIM-4, CD7, CD96,CD160, CD200, CD300a/LMIR1, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26,EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3,TIM-1/KIM-1/HAVCR, TIM-4, TSLP R, or any combinations thereof.

In the context of the present invention a checkpoint modulator isdefined herein as a molecule preferably a protein e.g. an antibody, adominant negative receptor, a decoy receptor, or a ligand or a fragmentor variant thereof, which modulates the function of an immune checkpointprotein, e.g. it inhibits or reduces the activity of checkpointinhibitors (or inhibitory checkpoint molecules) or it stimulates theactivity of checkpoint stimulators (or stimulatory checkpointmolecules). Therefore checkpoint modulators as defined herein, influencethe activity of checkpoint molecules.

In this context inhibitory checkpoint molecules are defined ascheckpoint inhibitors and can be used synonymously. In additionstimulatory checkpoint molecules are defined as checkpoint stimulatorsand can be used synonymously.

Preferable inhibitory checkpoint molecules that may be inhibited by acheckpoint modulator in the context of the invention are PD-1, PD-L1,CTLA-4, PD-L2, LAG3, TIM3/HAVCR2, 2B4, A2aR, B7H3, B7H4, BTLA, CD30,CD160, GAL9, HVEM, IDO1, IDO2, KIR, LAIR1 and VISTA.

Preferable stimulatory checkpoint molecules that may be stimulated by acheckpoint modulator in the context of the invention are CD2, CD27,CD28, CD40, CD137, CD226, CD276, GITR, ICOS, OX-40 and CD70.

Preferably, the checkpoint modulator is selected from agonisticantibodies, antagonistic antibodies, ligands, dominant negativereceptors, and decoy receptors or combinations thereof.

Methods for generating and using mRNA-encoded antibodies are known inthe art (e.g. WO2008/083949).

Preferably, the agonistic antibody is chosen from the following list:anti-4-1BB, anti-OX40, anti-GITR, anti-CD28, anti-CD27,anti-CD-40anti-ICOS, anti-TNFRSF25, and anti-LIGHT.

OX40 is a member of the TNFR-superfamily of receptors, and is expressedon the surface of antigen-activated mammalian CD4+ and CD8+ Tlymphocytes. OX40 ligand (OX40L, also known as gp34, ACT-4-L, and CD252)is a protein that specifically interacts with the OX40 receptor. Theterm OX40L includes the entire OX40 ligand, soluble OX40 ligand, andfusion proteins comprising a functionally active portion of OX40 ligandcovalently linked to a second moiety, e.g., a protein domain. Alsoincluded within the definition of OX40L are variants which vary in aminoacid sequence from naturally occurring OX4L but which retain the abilityto specifically bind to the OX40 receptor. Further included within thedefinition of OX40L are variants which enhance the biological activityof OX40. An OX40 agonist is a molecule which induces or enhances thebiological activity of OX40, e.g. signal transduction mediated by OX40.An OX40 agonist is preferably defined herein as a binding moleculecapable of specific binding to OX40. Therefore, the OX40 agonist may beany agonist binding to OX40 and capable of stimulating OX40 signaling.In this context, the OX40 agonist may be an agonistic antibody bindingto OX40.

OX40 agonists and anti-OX40 monoclonal antibodies are described inWO1995/021251, WO1995/012673 and WO1995/21915. Particularly preferred isthe anti-OX40 antibody 9B12, a murine anti-OX40 monoclonal antibodydirected against the extracellular domain of human OX40 (Weinberg etal., 2006. J. Immunother. 29(6):575-585).

Preferably, the antagonistic antibody is chosen from the list ofanti-CTLA4, anti-PD1, anti-PD-L1, anti-Vista, anti-Tim-3, anti-LAG-3,and anti-BTLA.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is mainly expressed within theintracellular compartment of T cells. After a potent or long-lastingstimulus to a naive T cell via the T cell receptor (TCR), CTLA-4 istransported to the cell surface and concentrated at the immunologicalsynapse. CTLA-4 then competes with CD28 for CD80/CD86 and down-modulatesTCR signaling via effects on Akt signaling. Thus CTLA-4 functionsphysiologically as a signal dampener (Weber, J. 2010. Semin. Oncol.37(5):430-9).

Particularly preferred are the anti-CTLA-4 antibodies ipilimumab(Yervoy®), tremelimumab, and AGEN-1884.

Members of the PD-1 pathway are all proteins which are associated withPD-1 signaling. On the one hand these might be proteins which inducePD-1 signaling upstream of PD-1 as e.g. the ligands of PD-1 PD-L1 andPD-L2 and the signal transduction receptor PD-1. On the other hand thesemight be signal transduction proteins downstream of PD-1 receptor.Particularly preferred as members of the PD-1 pathway in the context ofthe present invention are PD-1, PD-L1 and PD-L2.

In the context of the present invention, a PD-1 pathway antagonist ispreferably defined herein as a compound capable to impair the PD-1pathway signaling, preferably signaling mediated by the PD-1 receptor.Therefore, the PD-1 pathway antagonist may be any antagonist directedagainst any member of the PD-1 pathway capable of antagonizing PD-1pathway signaling. In this context, the antagonist may be anantagonistic antibody as defined herein, targeting any member of thePD-1 pathway, preferably directed against PD-1 receptor, PD-L1 or PD-L2.This antagonistic antibody may also be encoded by a nucleic acid. Also,the PD-1 pathway antagonist may be a fragment of the PD-1 receptorblocking the activity of PD1 ligands. B7-1 or fragments thereof may actas PD1-antagonizing ligands as well. Additionally, a PD-1 pathwayantagonist may be a protein comprising (or a nucleic acid coding for) anamino acid sequence capable of binding to PD-1 but preventing PD-1signaling, e.g. by inhibiting PD-1 and B7-H1 or B7-DL interaction(WO2014127917).

Particularly preferred are the anti-PD1 antibodies Nivolumab(MDX-1106/BMS-936558/ONO-4538), (Brahmer et al., 2010. J Clin Oncol.28(19):3167-75; PMID: 20516446); Pidilizumab (CT-011), (Berger et al.,2008. Clin Cancer Res. 14(10):3044-51; PMID: 18483370); Pembrolizumab(MK-3475, SCH 900475); AMP-224, and MED10680 (AMP-514)

Particularly preferred are the anti-PD-L1 antibodies MDX-1105/BMS-936559(Brahmer et al. 2012. N Engl J Med. 366(26):2455-65; PMID: 22658128);atezolizumab (MPDL3280A/RG7446); durvalumab (MED14736); and avelumab(MSB0010718).

According to the present invention the at least one RNA of the inventiveRNA containing composition encodes at least one antibody or fragments orvariants thereof of Table 10. It is particularly preferred that the RNAcontaining composition comprises at least one RNA encoding the heavychain of a particular antibody or fragments or variants thereof and atleast one further RNA encoding the light chain of the same particularantibody or fragments or variants thereof.

TABLE 10 Antibodies directed against checkpoint molecules Name TargetUrelumab 4-1BB/CD137 PF-05082566 4-1BB/CD137 8H9 B7-H3 EnoblituzumabB7-H3 Ipilimumab CD152/CTLA-4 Ticilimumab (=tremelimumab) CD152/CTLA-4Tremelimumab CD152/CTLA-4 Varlilumab CD27 Teneliximab CD40 Vorsetuzumabmafodotin CD70 Lirilumab KIR2D GSK-3174998 OX40 MEDI-6469 OX40 MEDI-6383OX40 MEDI-0562 OX40 PF-04518600 OX40 RG-7888 OX40 PF-06801591 PD-1BGBA-317 PD-1 MEDI-0680 PD-1 MK-3475 PD-1 Nivolumab PD-1 PDR-001 PD-1Pembrolizumab PD-1 Pidilizumab PD-1 REGN-2810 PD-1 SHR-1210 PD-1 TSR-042PD-1 MDX-1106 PD-1 Merck 3745 PD-1 CT-011 PD-1 MEDI-0680 PD-1 PDR001PD-1 REGN2810 PD-1 BGB-108 PD-1 BGB-A317 PD-1 AMP-224 PD-1 AtezolizumabPD-L1 (CD274) Avelumab PD-L1 (CD274) BMS-936559 PD-L1 (CD274) DurvalumabPD-L1 (CD274) MEDI-4736 PD-L1 (CD274) MPDL33280A PD-L1 (CD274)YW243.55.S70 PD-L1 (CD274) MDX-1105 PD-L1 (CD274) MSB0010718C PD-L1(CD274)

In a further preferred embodiment the checkpoint modulator is a decoyreceptor (e.g. a soluble receptor). Preferably, the decoy receptor is asoluble PD1 receptor. In a particularly preferred embodiment the atleast one RNA of the inventive RNA containing composition comprises anRNA sequence being identical or at least 50%, 60%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to the RNA sequence according to SEQ ID NO:389 encoding a soluble PD-1 receptor.

In a further preferred embodiment of the inventive RNA containingcomposition the RNA, preferably an mRNA codes for at least one ligandwhich functions as a checkpoint modulator. Preferably, the ligand isCD40 Ligand (CD40L). In a further preferred embodiment of the inventiveRNA containing composition the RNA, preferably an mRNA codes for atleast one ligand which functions as a checkpoint modulator. Preferably,the ligand is CD40 Ligand (CD40L). Most preferably the at least one RNAof the inventive RNA containing composition comprises an RNA sequencebeing identical or at least 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% identical to the RNA sequence according to SEQ ID NO: 10073 encodingCD40L.

12. Innate Immune Activators:

In this context innate immune activators may be selected from mammalian,in particular human adjuvant proteins, which typically comprise anyhuman protein or peptide, which is capable of eliciting an innate immuneresponse (in a mammal), e.g. as a reaction of the binding of anexogenous TLR ligand to a TLR. More preferably, human adjuvant proteinsare selected from the group consisting of proteins which are componentsand ligands of the signalling networks of the pattern recognitionreceptors including TLR, NLR and RLH, including TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4,NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7,NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, I IPAF, NAIP,CIITA, RIG-I, MDA5 and LGP2, the signal transducers of TLR signalingincluding adaptor proteins including e.g. Trif and Cardif; components ofthe Small-GTPases signalling (RhoA, Ras, Rac1, Cdc42, Rab etc.),components of the PIP signalling (PI3K, Src-Kinases, etc.), componentsof the MyD88-dependent signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP,TRAF6 etc.), components of the MyD88-independent signalling (TICAM1,TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinasesincluding e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2,GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK, p38MAPK, TAK1, IKK,and TAK1; the activated transcription factors including e.g. NF-κB,c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7.

Mammalian, in particular human adjuvant proteins may furthermore beselected from the group consisting of heat shock proteins, such asHSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIIIrepeat extra domain A of fibronectin; or components of the complementsystem including C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b,C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR,C1INH, C4bp, MCP, DAF, H, I, P and CD59, or induced target genesincluding e.g. Beta-Defensin, cell surface proteins; or human adjuvantproteins including trif, flt-3 ligand, Gp96 or fibronectin, etc., or anyspecies homolog of any of the above human adjuvant proteins. FurthermoreHGMB1 may be used as adjuvant protein.

Mammalian, in particular human adjuvant proteins may furthermorecomprise cytokines which induce or enhance an innate immune response,including IL-1 alpha, IL1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12,IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha,IFNbeta, IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8,IP-10, MCP-1, MIP-1alpha, RANTES, Eotaxin, CCL21; cytokines which arereleased from macrophages, including IL-1, IL-6, IL-8, IL-12 andTNF-alpha; as well as IL-1R1 and IL-1 alpha.

Therefore in this context it particularly preferred that the at leastone RNA encodes at least one innate immune activator, preferably anadjuvant protein, more preferably a human adjuvant protein, or afragment or variant thereof.

In this context it is particularly preferred that I constitutive activevariant of an adjuvant protein is encoded by the at least one RNA,preferably a constitutive active variant of RIG-1 (ΔRIGI).

In another preferred embodiment the at least one RNA encodes HGMB1 as aninnate immune activator, or a fragment or variant thereof.

According to preferred embodiments in the context of the presentinvention innate immune activators may be selected from any innateimmune activator selected from the group consisting of CD55; Akt; ATF2;C1QBP; C1QC; Cardif; CCL11; CCL2; CCL21; CCL3; CCL5; CD59,Beta-Defensin; Cdc42; CFAD; CFAH; CFAI; CH60; CIITA; c-Jun; c-myc; CO8A;COBB; CO8G; complement_system_component_C1INH;complement_system_component_C1qR; complement_system_component_C1s;complement_system_component_C4bp; complement_system_component_C6;complement_system_component_C7; complement_system_component_C8;complement_system_component_C9; complement_system_component_CR2;complement_system_component_CR3; complement_system_component_MASP-1;complement_system_component_MASP-2; complement_system_component_MBL;complement_system_component_MCP; CREB3; CREB3L1; CREB3L3; CREB3L4;CREB5; CRTC2; CXCL10; CXCL8; DJB11; DJB13; DJB14; DJC10; DJC12; DJC14;DJC15; DJC16; DJC17; DJC18; DJC22; DJC24; DJC25; DJC27; DJC28; DJC30;DNAJB12; DNAJC11; DNAJC21; DNJA1; DNJA2; DNJA3; DNJA4; DNJB1; DNJB2;DNJB3; DNJB4; DNJB5; DNJB6; DNJB7; DNJB8; DNJB9; DNJC1; DNJC2; DNJC3;DNJC4; DNJC5; DNJC7; DNJC8; DNJC9; Elk-1; ERK1; ERK2; Fibrinogen;fibronectin; FLT3_Ligand; FOS; G-CSF; GM-CSF; GRP94_(gp96); GSK3A;GSK3B; HS71A; HS71B; HSC70; HSP10; HSP60; HSP70; HSP75; HSP90; HSP90B1;IFNalpha; IFNB; IFNG; IKK; IL-1; IL-1_alpha; IL-1_beta; IL-12; IL-13;IL-15; IL-16; IL-17A; IL-18; IL-1R1; IL-2; IL-21; IL-23; IL-6; IL-7;IL-9; IRAK1; IRAK2; IRAK4; IRF3; IRF-7; JNK; KPCB; KPCD; KPCD1; KPCD3;KPCE; KPCG; KPCI; KPCL; KPCT; KPCZ; I_IPAF; LGP2; M-CSF; MDA5; MK11;MK12; MK13; MK14; MKK1; MKK3; MKK4; MKK6; MKK7; MSTP104; MyD88; NALP10;NALP11; NALP12; NALP13; NALP2; NALP3; NALP4; NALP5; NALP6; NALP7; NALP8;NALP9; NF-kappaB; NLRP14; NOD1; NOD2; NOD3; PI3K; PKD2; PKN1; PKN2;PKN3; PRKCA; PRKD2; Rab; Rac1; RASH; RASK; RASN; RhoA; RIG-I;Src-Kinases; Surfactant_protein_A; Surfactant_protein_D; TAK1; TBK1;TICAM1; TICAM2; TIRAP; TLR1; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7;TLR8; TLR9; TNF; TRAF6, preferably as disclosed in Table 11.Particularly preferred in this context are the RNA sequences encoding ainnate immune activator according to Table 11.

TABLE 11 Innate immune activators (human adjuvant proteins) RNA ProteinSequence Sequence wild type Protein SEQ ID SEQ ID Optimized RNA SequenceGene Name Accession No. NO: NO: SEQ ID NO: CD55 UniProtKB: 909 910 911,912, 913, 914, 915 B1AP15 CD55 UniProtKB: 916 917 918, 919, 920, 921,922 D3DT85 CD55 UniProtKB: 923 924 925, 926, 927, 928, 929 D3DT86 CD55UniProtKB: 930 931 932, 933, 934, 935, 936 P08174 fibronectin UniProtKB:1567 1568 1569, 1570, 1571, 1572, 1573 A0A024R516 fibronectin UniProtKB:1574 1575 1576, 1577, 1578, 1579, 1580 A0A024111301 fibronectinUniProtKB: 1581 1582 1583, 1584, 1585, 1586, 1587 A0A024RDT9 fibronectinUniProtKB: 1588 1589 1590, 1591, 1592, 1593, 1594 A0A024RDV5 fibronectinUniProtKB: 1595 1596 1597, 1598, 1599, 1600, 1601 A6NH44 fibronectinUniProtKB: 1602 1603 1604, 1605, 1606, 1607, 1608 A8K6A5 fibronectinUniProtKB: 1609 1610 1611, 1612, 1613, 1614, 1615 B2R627 fibronectinUniProtKB: 1616 1617 1618, 1619, 1620, 1621, 1622 B3KXM5 fibronectinUniProtKB: 1623 1624 1625, 1626, 1627, 1628, 1629 B4DIC5 fibronectinUniProtKB: 1630 1631 1632, 1633, 1634, 1635, 1636 B4DN21 fibronectinUniProtKB: 1637 1638 1639, 1640, 1641, 1642, 1643 B4DS98 fibronectinUniProtKB: 1644 1645 1646, 1647, 1648, 1649, 1650 B4DTH2 fibronectinUniProtKB: 1651 1652 1653, 1654, 1655, 1656, 1657 B4DTK1 fibronectinUniProtKB: 1658 1659 1660, 1661, 1662, 1663, 1664 B4DU16 fibronectinUniProtKB: 1665 1666 1667, 1668, 1669, 1670, 1671 B7Z3W5 fibronectinUniProtKB: 1672 1673 1674, 1675, 1676, 1677, 1678 B7Z939 fibronectinUniProtKB: 1679 1680 1681, 1682, 1683, 1684, 1685 G5E9X3 fibronectinUniProtKB: 1686 1687 1688, 1689, 1690, 1691, 1692 Q9H382 FOS UniProtKB:1693 1694 1695, 1696, 1697, 1698, 1699 P01100 HS71A UniProtKB: 1938 19391940, 1941, 1942, 1943, 1944 P0DMV8 HS71B UniProtKB: 1945 1946 1947,1948, 1949, 1950, 1951 P0DMV9 RASH UniProtKB: 3296 3297 3298, 3299,3300, 3301, 3302 P01112 RASK UniProtKB: 3303 3304 3305, 3306, 3307,3308, 3309 P01116 RASN UniProtKB: 3310 3311 3312, 3313, 3314, 3315, 3316P01111 FLT3 Jigand Genbank: 3913 3914 3915, 3916, 3917, 3918, 3919,AAA90950.1 3920 FLT3 Jigand UniProtKB: 3921 3922 3923, 3924, 3925, 3926,3927, P49771 3928 G-CSF UniProtKB: 3929 3930 3931, 3932, 3933, 3934,3935, P09919 3936 GM-CSF UniProtKB: 3945 3946 3947, 3948, 3949, 3950,3951, P04141 3952 IFNalpha UniProtKB: 3953 3954 3955, 3956, 3957, 3958,3959, G9JKF1 3960 IFNalpha UniProtKB: 3961 3962 3963, 3964, 3965, 3966,3967, P01562 3968 IFNalpha UniProtKB: 3969 3970 3971, 3972, 3973, 3974,3975, P01563 3976 IFNalpha UniProtKB: 3977 3978 3979, 3980, 3981, 3982,3983, P01566 3984 IFNalpha UniProtKB: 3985 3986 3987, 3988, 3989, 3990,3991, P01567 3992 IFNalpha UniProtKB: 3993 3994 3995, 3996, 3997, 3998,3999, P01568 4000 IFNalpha UniProtKB: 4001 4002 4003, 4004, 4005, 4006,4007, P01569 4008 IFNalpha UniProtKB: 4009 4010 4011, 4012, 4013, 4014,4015, P01570 4016 IFNalpha UniProtKB: 4017 4018 4019, 4020, 4021, 4022,4023, P01571 4024 IFNalpha UniProtKB: 4025 4026 4027, 4028, 4029, 4030,4031, P05013 4032 IFNalpha UniProtKB: 4033 4034 4035, 4036, 4037, 4038,4039, P05014 4040 IFNalpha UniProtKB: 4041 4042 4043, 4044, 4045, 4046,4047, P05015 4048 IFNalpha UniProtKB: 4049 4050 4051, 4052, 4053, 4054,4055, P32881 4056 IFNalpha UniProtKB: 4057 4058 4059, 4060, 4061, 4062,4063, Q14618 4064 IFNalpha UniProtKB: 4065 4066 4067, 4068, 4069, 4070,4071, Q86UP4 4072 IFNB UniProtKB: 4073 4074 4075, 4076, 4077, 4078,4079, P01574 4080 IFNB UniProtKB: 4081 4082 4083, 4084, 4085, 4086,4087, Q15943 4088 IFNG UniProtKB: 4089 4090 4091, 4092, 4093, 4094,4095, P01579 4096 IFNG UniProtKB: 4097 4098 4099, 4100, 4101, 4102,4103, Q14609 4104 IFNG UniProtKB: 4105 4106 4107, 4108, 4109, 4110,4111, Q14610 4112 IFNG UniProtKB: 4113 4114 4115, 4116, 4117, 4118,4119, Q14611 4120 IFNG UniProtKB: 4121 4122 4123, 4124, 4125, 4126,4127, Q14612 4128 IFNG UniProtKB: 4129 4130 4131, 4132, 4133, 4134,4135, Q14613 4136 IFNG UniProtKB: 4137 4138 4139, 4140, 4141, 4142,4143, Q14614 4144 IFNG UniProtKB: 4145 4146 4147, 4148, 4149, 4150,4151, Q14615 4152 IFNG UniProtKB: 4153 4154 4155, 4156, 4157, 4158,4159, Q8NHY9 4160 IL-12 UniProtKB: 4193 4194 4195, 4196, 4197, 4198,4199, P29460 4200 IL-13 UniProtKB: 4201 4202 4203, 4204, 4205, 4206,4207, P35225 4208 IL-15 UniProtKB: 4217 4218 4219, 4220, 4221, 4222,4223, P40933 4224 IL-16 UniProtKB: 4225 4226 4227, 4228, 4229, 4230,4231, Q14005 4232 IL-17A UniProtKB: 4233 4234 4235, 4236, 4237, 4238,4239, Q16552 4240 IL-18 UniProtKB: 4281 4282 4283, 4284, 4285, 4286,4287, A0A024113E0 4288 IL-18 UniProtKB: 4289 4290 4291, 4292, 4293,4294, 4295, B0YJ28 4296 IL-18 UniProtKB: 4297 4298 4299, 4300, 4301,4302, 4303, Q14116 4304 IL-1_alpha UniProtKB: 4313 4314 4315, 4316,4317, 4318, 4319, P01583 4320 IL-1_beta UniProtKB: 4321 4322 4323, 4324,4325, 4326, 4327, P01584 4328 IL-21 RefSeq: 4361 4362 4363, 4364, 4365,4366, 4367, NP_001193935.1 4368 IL-21 RefSeq: 4369 4370 4371, 4372,4373, 4374, 4375, NP_068575.1 4376 IL-23 UniProtKB: 4385 4386 4387,4388, 4389, 4390, 4391, Q9NPF7 4392 IL-2 UniProtKB: 4473 4474 4475,4476, 4477, 4478, 4479, P60568 4480 IL-2 UniProtKB: 4481 4482 4483,4484, 4485, 4486, 4487, Q0GK43 4488 IL-2 UniProtKB: 4489 4490 4491,4492, 4493, 4494, 4495, Q13169 4496 IL-2 UniProtKB: 4497 4498 4499,4500, 4501, 4502, 4503, Q6NZ91 4504 IL-2 UniProtKB: 4505 4506 4507,4508, 4509, 4510, 4511, Q6NZ93 4512 IL-6 UniProtKB: 4593 4594 4595,4596, 4597, 4598, 4599, P05231 4600 IL-7 UniProtKB: 4601 4602 4603,4604, 4605, 4606, 4607, A8K673 4608 IL-7 UniProtKB: 4609 4610 4611,4612, 4613, 4614, 4615, P13232 4616 IL-9 UniProtKB: 4617 4618 4619,4620, 4621, 4622, 4623, P15248 4624 M-CSF UniProtKB: 4705 4706 4707,4708, 4709, 4710, 4711, P09603 4712 CCL11 UniProtKB: 4833 4834 4835,4836, 4837, 4838, 4839, P51671 4840 CCL11 UniProtKB: 4841 4842 4843,4844, 4845, 4846, 4847, Q619T4 4848 CCL21 UniProtKB: 4937 4938 4939,4940, 4941, 4942, 4943, O00585 4944 CCL2 UniProtKB: 5001 5002 5003,5004, 5005, 5006, 5007, P13500 5008 CCL3 UniProtKB: 5009 5010 5011,5012, 5013, 5014, 5015, A0N0R1 5016 CCL3 UniProtKB: 5017 5018 5019,5020, 5021, 5022, 5023, P10147 5024 CCL5 UniProtKB: 5041 5042 5043,5044, 5045, 5046, 5047, D0EI67 5048 CCL5 UniProtKB: 5049 5050 5051,5052, 5053, 5054, 5055, P13501 5056 CXCL10 UniProtKB: 5129 5130 5131,5132, 5133, 5134, 5135, A0A024RDA4 5136 CXCL10 UniProtKB: 5137 51385139, 5140, 5141, 5142, 5143, P02778 5144 CXCL8 UniProtKB: 5265 52665267, 5268, 5269, 5270, 5271, P10145 5272 TNF UniProtKB: 7369 7370 7371,7372, 7373, 7374, 7375, P01375 7376 TNF UniProtKB: 7377 7378 7379, 7380,7381, 7382, 7383, Q5STB3 7384 GRP94_(gp96) UniProtKB: 7617 7618 7619,7620, 7621, 7622, 7623, P14625 7624 HSC70 UniProtKB: 7625 7626 7627,7628, 7629, 7630, 7631, P11142 7632 HSP60 UniProtKB: 7657 7658 7659,7660, 7661, 7662, 7663, A0A024R3X4 7664 HSP60 UniProtKB: 7665 7666 7667,7668, 7669, 7670, 7671, B3GQS7 7672 HSP60 UniProtKB: 7673 7674 7675,7676, 7677, 7678, 7679, P10809 7680 HSP60 UniProtKB: 7681 7682 7683,7684, 7685, 7686, 7687, Q0VDF9 7688 HSP70 UniProtKB: 7689 7690 7691,7692, 7693, 7694, 7695, P38646 7696 HSP90 UniProtKB: 7697 7698 7699,7700, 7701, 7702, 7703, P07900 7704 HSP90 UniProtKB: 7705 7706 7707,7708, 7709, 7710, 7711, P08238 7712 Akt UniProtKB: 7737 7738 7739, 7740,7741, 7742, 7743 B0LPE5 Akt UniProtKB: 7744 7745 7746, 7747, 7748, 7749,7750 P31749 Akt UniProtKB: 7751 7752 7753, 7754, 7755, 7756, 7757 P31751Akt UniProtKB: 7758 7759 7760, 7761, 7762, 7763, 7764 Q9Y243 ATF2UniProtKB: 7765 7766 7767, 7768, 7769, 7770, 7771 P15336 C1QBPUniProtKB: 7772 7773 7774, 7775, 7776, 7777, 7778 Q07021 C1QC UniProtKB:7779 7780 7781, 7782, 7783, 7784, 7785 P02747 Cardif UniProtKB: 77867787 7788, 7789, 7790, 7791, 7792 Q72434 CD59, Beta-Defensin UniProtKB:7793 7794 7795, 7796, 7797, 7798, 7799 P13987 CD59, Beta-DefensinUniProtKB: 7800 7801 7802, 7803, 7804, 7805, 7806 Q6FHM9 Cdc42UniProtKB: 7807 7808 7809, 7810, 7811, 7812, 7813 A0A024RAE4 Cdc42UniProtKB: 7814 7815 7816, 7817, 7818, 7819, 7820 A0A024RAE6 Cdc42UniProtKB: 7821 7822 7823, 7824, 7825, 7826, 7827 P60953 CFAD UniProtKB:7828 7829 7830, 7831, 7832, 7833, 7834 P00746 CFAH UniProtKB: 7835 78367837, 7838, 7839, 7840, 7841 P08603 CFAI UniProtKB: 7842 7843 7844,7845, 7846, 7847, 7848 P05156 CH60 RefSeq: 7849 7850 7851, 7852, 7853,7854, 7855 NP_002147.2 CIITA UniProtKB: 7856 7857 7858, 7859, 7860,7861, 7862 Q29704 c-Jun UniProtKB: 7863 7864 7865, 7866, 7867, 7868,7869 B3KN68 c-Jun UniProtKB: 7870 7871 7872, 7873, 7874, 7875, 7876B3KNW1 c-Jun UniProtKB: 7877 7878 7879, 7880, 7881, 7882, 7883 B3KXW5c-Jun UniProtKB: 7884 7885 7886, 7887, 7888, 7889, 7890 B4DED9 c-JunUniProtKB: 7891 7892 7893, 7894, 7895, 7896, 7897 B4DFU7 c-JunUniProtKB: 7898 7899 7900, 7901, 7902, 7903, 7904 B4DGE1 c-JunUniProtKB: 7905 7906 7907, 7908, 7909, 7910, 7911 B4DJ64 c-JunUniProtKB: 7912 7913 7914, 7915, 7916, 7917, 7918 B4DS36 c-JunUniProtKB: 7919 7920 7921, 7922, 7923, 7924, 7925 B7Z1L7 c-JunUniProtKB: 7926 7927 7928, 7929, 7930, 7931, 7932 G1U124 c-JunUniProtKB: 7933 7934 7935, 7936, 7937, 7938, 7939 G5E966 c-JunUniProtKB: 7940 7941 7942, 7943, 7944, 7945, 7946 O75843 c-JunUniProtKB: 7947 7948 7949, 7950, 7951, 7952, 7953 P05412 c-JunUniProtKB: 7954 7955 7956, 7957, 7958, 7959, 7960 P53677 c-JunUniProtKB: 7961 7962 7963, 7964, 7965, 7966, 7967 P61966 c-JunUniProtKB: 7968 7969 7970, 7971, 7972, 7973, 7974 Q63HQ0 c-JunUniProtKB: 7975 7976 7977, 7978, 7979, 7980, 7981 Q7Z5Q8 c-JunUniProtKB: 7982 7983 7984, 7985, 7986, 7987, 7988 Q96PC3 c-JunUniProtKB: 7989 7990 7991, 7992, 7993, 7994, 7995 Q9BXS5 c-JunUniProtKB: 7996 7997 7998, 7999, 8000, 8001, 8002 Q9Y6Q5 CO8A UniProtKB:8003 8004 8005, 8006, 8007, 8008, 8009 P07357 COBB UniProtKB: 8010 80118012, 8013, 8014, 8015, 8016 P07358 CO8G UniProtKB: 8017 8018 8019,8020, 8021, 8022, 8023 P07360 complement_system_ UniProtKB: 8024 80258026, 8027, 8028, 8029, 8030 component_C1lNH P05155 complement_system_UniProtKB: 8031 8032 8033, 8034, 8035, 8036, 8037 component_C1q11 Q8IXK1complement_system_ UniProtKB: 8038 8039 8040, 8041, 8042, 8043, 8044component_C1s P09871 complement_system_ UniProtKB: 8045 8046 8047, 8048,8049, 8050, 8051 component_C4bp P04003 complement_system_ UniProtKB:8052 8053 8054, 8055, 8056, 8057, 8058 component_C6 P13671complement_system_ UniProtKB: 8059 8060 8061, 8062, 8063, 8064, 8065component_C7 P10643 complement_system_ UniProtKB: 8066 8067 8068, 8069,8070, 8071, 8072 component_C8 Q99618 complement_system_ UniProtKB: 80738074 8075, 8076, 8077, 8078, 8079 component_C9 A0A024R035complement_system_ UniProtKB: 8080 8081 8082, 8083, 8084, 8085, 8086component_C9 P02748 complement_system_ UniProtKB: 8087 8088 8089, 8090,8091, 8092, 8093 component_CR2 P20023 complement_system_ UniProtKB: 80948095 8096, 8097, 8098, 8099, 8100 component_CR3 D3DSM0complement_system_ UniProtKB: 8101 8102 8103, 8104, 8105, 8106, 8107component_CR3 P05107 complement_system_ UniProtKB: 8108 8109 8110, 8111,8112, 8113, 8114 component_MASP-1 P48740 complement_system_ UniProtKB:8115 8116 8117, 8118, 8119, 8120, 8121 component_MASP-2 000187complement_system_ UniProtKB: 8122 8123 8124, 8125, 8126, 8127, 8128component_MBL P11226 complement_system_ UniProtKB: 8129 8130 8131, 8132,8133, 8134, 8135 component_MCP P15529 complement_system_ UniProtKB: 81368137 8138, 8139, 8140, 8141, 8142 component_MCP P40121 CREB3 CCDS: 81438144 8145, 8146, 8147, 8148, 8149 CCDS6588.1 CREB3L1 UniProtKB: 81508151 8152, 8153, 8154, 8155, 8156 Q96BA8 CREB3L3 UniProtKB: 8157 81588159, 8160, 8161, 8162, 8163 Q68CJ9 CREB3L4 UniProtKB: 8164 8165 8166,8167, 8168, 8169, 8170 Q8TEY5 CREBS UniProtKB: 8171 8172 8173, 8174,8175, 8176, 8177 Q02930 CRTC2 UniProtKB: 8178 8179 8180, 8181, 8182,8183, 8184 Q53ET0 DJB11 UniProtKB: 8185 8186 8187, 8188, 8189, 8190,8191 Q9UBS4 DJB13 UniProtKB: 8192 8193 8194, 8195, 8196, 8197, 8198P59910 DJB14 UniProtKB: 8199 8200 8201, 8202, 8203, 8204, 8205 Q8TBM8DJC10 UniProtKB: 8206 8207 8208, 8209, 8210, 8211, 8212 Q81X81 DJC12UniProtKB: 8213 8214 8215, 8216, 8217, 8218, 8219 Q9UKB3 DJC14UniProtKB: 8220 8221 8222, 8223, 8224, 8225, 8226 Q6Y2X3 DJC15UniProtKB: 8227 8228 8229, 8230, 8231, 8232, 8233 Q9Y5T4 DJC16UniProtKB: 8234 8235 8236, 8237, 8238, 8239, 8240 Q9Y2G8 DJC17UniProtKB: 8241 8242 8243, 8244, 8245, 8246, 8247 Q9NVM6 DJC18UniProtKB: 8248 8249 8250, 8251, 8252, 8253, 8254 Q9H819 DJC22UniProtKB: 8255 8256 8257, 8258, 8259, 8260, 8261 Q8N4W6 DJC24UniProtKB: 8262 8263 8264, 8265, 8266, 8267, 8268 Q6P3W2 DJC25UniProtKB: 8269 8270 8271, 8272, 8273, 8274, 8275 Q9H1X3 DJC27UniProtKB: 8276 8277 8278, 8279, 8280, 8281, 8282 Q9NZQ0 DJC28UniProtKB: 8283 8284 8285, 8286, 8287, 8288, 8289 Q9NX36 DJC30UniProtKB: 8290 8291 8292, 8293, 8294, 8295, 8296 Q96LL9 DNAJB12 RefSeq:8297 8298 8299, 8300, 8301, 8302, 8303 NP_001002762.2 DNAJC11 UniProtKB:8304 8305 8306, 8307, 8308, 8309, 8310 Q9NVH1 DNAJC21 UniProtKB: 83118312 8313, 8314, 8315, 8316, 8317 Q5F1R6 DNJA1 UniProtKB: 8318 83198320, 8321, 8322, 8323, 8324 P31689 DNJA2 UniProtKB: 8325 8326 8327,8328, 8329, 8330, 8331 O60884 DNJA3 UniProtKB: 8332 8333 8334, 8335,8336, 8337, 8338 Q96EY1 DNJA4 UniProtKB: 8339 8340 8341, 8342, 8343,8344, 8345 Q8WW22 DNJB1 UniProtKB: 8346 8347 8348, 8349, 8350, 8351,8352 P25685 DNJB2 UniProtKB: 8353 8354 8355, 8356, 8357, 8358, 8359P25686 DNJB3 UniProtKB: 8360 8361 8362, 8363, 8364, 8365, 8366 Q8WWF6DNJB4 UniProtKB: 8367 8368 8369, 8370, 8371, 8372, 8373 Q9UDY4 DNJB5UniProtKB: 8374 8375 8376, 8377, 8378, 8379, 8380 O75953 DNJB6UniProtKB: 8381 8382 8383, 8384, 8385, 8386, 8387 O75190 DNJB7UniProtKB: 8388 8389 8390, 8391, 8392, 8393, 8394 Q7Z6W7 DNJB8UniProtKB: 8395 8396 8397, 8398, 8399, 8400, 8401 Q8NHS0 DNJB9UniProtKB: 8402 8403 8404, 8405, 8406, 8407, 8408 Q9UBS3 DNJC1UniProtKB: 8409 8410 8411, 8412, 8413, 8414, 8415 Q96KC8 DNJC2UniProtKB: 8416 8417 8418, 8419, 8420, 8421, 8422 Q99543 DNJC3UniProtKB: 8423 8424 8425, 8426, 8427, 8428, 8429 Q13217 DNJC4UniProtKB: 8430 8431 8432, 8433, 8434, 8435, 8436 Q9NNZ3 DNJCSUniProtKB: 8437 8438 8439, 8440, 8441, 8442, 8443 Q9H3Z4 DNJC7UniProtKB: 8444 8445 8446, 8447, 8448, 8449, 8450 Q99615 DNJC8UniProtKB: 8451 8452 8453, 8454, 8455, 8456, 8457 O75937 DNJC9UniProtKB: 8458 8459 8460, 8461, 8462, 8463, 8464 Q8WXX5 Elk-1UniProtKB: 8465 8466 8467, 8468, 8469, 8470, 8471 P19419 Elk-1UniProtKB: 8472 8473 8474, 8475, 8476, 8477, 8478 Q8N9S0 ERK1 UniProtKB:8479 8480 8481, 8482, 8483, 8484, 8485 P27361 ERK2 UniProtKB: 8486 84878488, 8489, 8490, 8491, 8492 P28482 Fibrinogen UniProtKB: 8493 84948495, 8496, 8497, 8498, 8499 A0A024R8B4 Fibrinogen UniProtKB: 8500 85018502, 8503, 8504, 8505, 8506 A4D1B8 Fibrinogen UniProtKB: 8507 85088509, 8510, 8511, 8512, 8513 A8K8X4 Fibrinogen UniProtKB: 8514 85158516, 8517, 8518, 8519, 8520 B4DTN2 Fibrinogen UniProtKB: 8521 85228523, 8524, 8525, 8526, 8527 B4E1D3 Fibrinogen UniProtKB: 8528 85298530, 8531, 8532, 8533, 8534 D3DP13 Fibrinogen UniProtKB: 8535 85368537, 8538, 8539, 8540, 8541 D3DP16 Fibrinogen UniProtKB: 8542 85438544, 8545, 8546, 8547, 8548 D3DSP9 Fibrinogen UniProtKB: 8549 85508551, 8552, 8553, 8554, 8555 P02671 Fibrinogen UniProtKB: 8556 85578558, 8559, 8560, 8561, 8562 P02675 Fibrinogen UniProtKB: 8563 85648565, 8566, 8567, 8568, 8569 P02679 Fibrinogen UniProtKB: 8570 85718572, 8573, 8574, 8575, 8576 Q08830 Fibrinogen UniProtKB: 8577 85788579, 8580, 8581, 8582, 8583 Q14314 Fibrinogen UniProtKB: 8584 85858586, 8587, 8588, 8589, 8590 Q6UXM4 Fibrinogen UniProtKB: 8591 85928593, 8594, 8595, 8596, 8597 Q9UE34 FOS UniProtKB: 8598 8599 8600, 8601,8602, 8603, 8604 A0A024RD16 GSK3A UniProtKB: 8605 8606 8607, 8608, 8609,8610, 8611 P49840 GSK3B UniProtKB: 8612 8613 8614, 8615, 8616, 8617,8618 P49841 HSP10 UniProtKB: 8619 8620 8621, 8622, 8623, 8624, 8625P61604 HSP75 UniProtKB: 8626 8627 8628, 8629, 8630, 8631, 8632 Q12931HSP90B1 UniProtKB: 8633 8634 8635, 8636, 8637, 8638, 8639 Q5CAQ5 IKKUniProtKB: 8640 8641 8642, 8643, 8644, 8645, 8646 O14920 IKK UniProtKB:8647 8648 8649, 8650, 8651, 8652, 8653 Q14164 IKK UniProtKB: 8654 86558656, 8657, 8658, 8659, 8660 Q9Y6K9 IL-1 UniProtKB: 8661 8662 8663,8664, 8665, 8666, 8667 O43353 IL-1 UniProtKB: 8668 8669 8670, 8671,8672, 8673, 8674 Q8N9C1 IL-1 UniProtKB: 8675 8676 8677, 8678, 8679,8680, 8681 Q8WWZ1 IL-1 UniProtKB: 8682 8683 8684, 8685, 8686, 8687, 8688Q9NZH7 IL-1 UniProtKB: 8689 8690 8691, 8692, 8693, 8694, 8695 Q9UBH0IL-1 UniProtKB: 8696 8697 8698, 8699, 8700, 8701, 8702 Q9UHA7 IL-1R1UniProtKB: 8703 8704 8705, 8706, 8707, 8708, 8709 P14778 IL-1R1UniProtKB: 8710 8711 8712, 8713, 8714, 8715, 8716 Q6NWP5 IL-1R1UniProtKB: 8717 8718 8719, 8720, 8721, 8722, 8723 Q6NWP6 IRAK1UniProtKB: 8724 8725 8726, 8727, 8728, 8729, 8730 L8E7M9 IRAK1UniProtKB: 8731 8732 8733, 8734, 8735, 8736, 8737 P51617 IRAK2UniProtKB: 8738 8739 8740, 8741, 8742, 8743, 8744 O43187 IRAK4UniProtKB: 8745 8746 8747, 8748, 8749, 8750, 8751 Q69FE3 IRAK4UniProtKB: 8752 8753 8754, 8755, 8756, 8757, 8758 Q7Z6A7 IRAK4UniProtKB: 8759 8760 8761, 8762, 8763, 8764, 8765 Q7Z6A8 IRAK4UniProtKB: 8766 8767 8768, 8769, 8770, 8771, 8772 Q9NWZ3 IRF3 UniProtKB:8773 8774 8775, 8776, 8777, 8778, 8779 A0A024QZE1 IRF3 UniProtKB: 87808781 8782, 8783, 8784, 8785, 8786 E2GIM5 IRF3 UniProtKB: 8787 8788 8789,8790, 8791, 8792, 8793 E2GIM6 IRF3 UniProtKB: 8794 8795 8796, 8797,8798, 8799, 8800 E2GIM7 IRF3 UniProtKB: 8801 8802 8803, 8804, 8805,8806, 8807 E2GIM8 IRF3 UniProtKB: 8808 8809 8810, 8811, 8812, 8813, 8814E2GIM9 IRF3 UniProtKB: 8815 8816 8817, 8818, 8819, 8820, 8821 Q14653IRF3 UniProtKB: 8822 8823 8824, 8825, 8826, 8827, 8828 Q96GL3 IRF-7UniProtKB: 8829 8830 8831, 8832, 8833, 8834, 8835 Q92985 JNK UniProtKB:8836 8837 8838, 8839, 8840, 8841, 8842 B4DU99 KPCB UniProtKB: 8843 88448845, 8846, 8847, 8848, 8849 P05771-1 KPCB UniProtKB: 8850 8851 8852,8853, 8854, 8855, 8856 P05771-2 KPCD1 UniProtKB: 8857 8858 8859, 8860,8861, 8862, 8863 Q15139 KPCD3 UniProtKB: 8864 8865 8866, 8867, 8868,8869, 8870 O94806 KPCD UniProtKB: 8871 8872 8873, 8874, 8875, 8876, 8877Q05655 KPCE UniProtKB: 8878 8879 8880, 8881, 8882, 8883, 8884 Q02156KPCG UniProtKB: 8885 8886 8887, 8888, 8889, 8890, 8891 P05129 KPCIUniProtKB: 8892 8893 8894, 8895, 8896, 8897, 8898 P41743 KPCL UniProtKB:8899 8900 8901, 8902, 8903, 8904, 8905 P24723 KPCT UniProtKB: 8906 89078908, 8909, 8910, 8911, 8912 Q04759 KPCZ UniProtKB: 8913 8914 8915,8916, 8917, 8918, 8919 Q05513 LGP2 UniProtKB: 8920 8921 8922, 8923,8924, 8925, 8926 A0A024R1Y5 LGP2 UniProtKB: 8927 8928 8929, 8930, 8931,8932, 8933 Q96C10 I_IPAF UniProtKB: 8934 8935 8936, 8937, 8938, 8939,8940 Q9NPP4 MDA5 UniProtKB: 8941 8942 8943, 8944, 8945, 8946, 8947Q9BYX4 MK11 UniProtKB: 8948 8949 8950, 8951, 8952, 8953, 8954 Q15759MK12 UniProtKB: 8955 8956 8957, 8958, 8959, 8960, 8961 P53778 MK13UniProtKB: 8962 8963 8964, 8965, 8966, 8967, 8968 O15264 MK14 UniProtKB:8969 8970 8971, 8972, 8973, 8974, 8975 Q16539 MKK1 UniProtKB: 8976 89778978, 8979, 8980, 8981, 8982 Q02750 MKK3 UniProtKB: 8983 8984 8985,8986, 8987, 8988, 8989 P46734 MKK4 UniProtKB: 8990 8991 8992, 8993,8994, 8995, 8996 P45985 MKK6 UniProtKB: 8997 8998 8999, 9000, 9001,9002, 9003 P52564 MKK7 UniProtKB: 9004 9005 9006, 9007, 9008, 9009, 9010O14733 MSTP104 UniProtKB: 9011 9012 9013, 9014, 9015, 9016, 9017 Q7Z4D5MyD88 UniProtKB: 9018 9019 9020, 9021, 9022, 9023, 9024 Q99836 NALP10UniProtKB: 9025 9026 9027, 9028, 9029, 9030, 9031 Q86W26 NALP11UniProtKB: 9032 9033 9034, 9035, 9036, 9037, 9038 P59045 NALP12UniProtKB: 9039 9040 9041, 9042, 9043, 9044, 9045 P59046 NALP13UniProtKB: 9046 9047 9048, 9049, 9050, 9051, 9052 Q86W25 NALP2UniProtKB: 9053 9054 9055, 9056, 9057, 9058, 9059 Q8WY49 NALP2UniProtKB: 9060 9061 9062, 9063, 9064, 9065, 9066 Q9NX02 NALP3UniProtKB: 9067 9068 9069, 9070, 9071, 9072, 9073 Q96P20 NALP4UniProtKB: 9074 9075 9076, 9077, 9078, 9079, 9080 Q96MN2 NALP5UniProtKB: 9081 9082 9083, 9084, 9085, 9086, 9087 P59047 NALP6UniProtKB: 9088 9089 9090, 9091, 9092, 9093, 9094 P59044 NALP7UniProtKB: 9095 9096 9097, 9098, 9099, 9100, 9101 Q8WX94 NALP8UniProtKB: 9102 9103 9104, 9105, 9106, 9107, 9108 Q86W28 NALP9UniProtKB: 9109 9110 9111, 9112, 9113, 9114, 9115 Q7RTR0 NF-kappaBUniProtKB: 9116 9117 9118, 9119, 9120, 9121, 9122 A3F768 NF-kappaBUniProtKB: 9123 9124 9125, 9126, 9127, 9128, 9129 A3F769 NLRP14UniProtKB: 9130 9131 9132, 9133, 9134, 9135, 9136 Q86UT6 NLRP14UniProtKB: 9137 9138 9139, 9140, 9141, 9142, 9143 Q86W24 NOD1 UniProtKB:9144 9145 9146, 9147, 9148, 9149, 9150 G3XAL1 NOD1 UniProtKB: 9151 91529153, 9154, 9155, 9156, 9157 Q9Y239 NOD2 UniProtKB: 9158 9159 9160,9161, 9162, 9163, 9164 Q9HC29 NOD3 UniProtKB: 9165 9166 9167, 9168,9169, 9170, 9171 C3VPR7 NOD3 UniProtKB: 9172 9173 9174, 9175, 9176,9177, 9178 H3BLT9 NOD3 UniProtKB: 9179 9180 9181, 9182, 9183, 9184, 9185Q7RTR2 P13K UniProtKB: 9186 9187 9188, 9189, 9190, 9191, 9192 O00329P13K UniProtKB: 9193 9194 9195, 9196, 9197, 9198, 9199 O00459 P13KUniProtKB: 9200 9201 9202, 9203, 9204, 9205, 9206 P27986 P13K UniProtKB:9207 9208 9209, 9210, 9211, 9212, 9213 P42336 P13K UniProtKB: 9214 92159216, 9217, 9218, 9219, 9220 P42338 P13K UniProtKB: 9221 9222 9223,9224, 9225, 9226, 9227 P48736 P13K UniProtKB: 9228 9229 9230, 9231,9232, 9233, 9234 Q5UE93 P13K UniProtKB: 9235 9236 9237, 9238, 9239,9240, 9241 Q8NEB9 P13K UniProtKB: 9242 9243 9244, 9245, 9246, 9247, 9248Q8WYR1 PKD2 UniProtKB: 9249 9250 9251, 9252, 9253, 9254, 9255 Q13563PKN1 UniProtKB: 9256 9257 9258, 9259, 9260, 9261, 9262 Q16512 PKN2UniProtKB: 9263 9264 9265, 9266, 9267, 9268, 9269 Q16513 PKN3 UniProtKB:9270 9271 9272, 9273, 9274, 9275, 9276 Q6P5Z2 PRKCA UniProtKB: 9277 92789279, 9280, 9281, 9282, 9283 P17252 PRKD2 RefSeq: 9284 9285 9286, 9287,9288, 9289, 9290 NP_001073349.1 Rab UniProtKB: 9291 9292 9293, 9294,9295, 9296, 9297 P52594 Rac1 UniProtKB: 9298 9299 9300, 9301, 9302,9303, 9304 A4D2P0 Rac1 UniProtKB: 9305 9306 9307, 9308, 9309, 9310, 9311A4D2P1 Rac1 UniProtKB: 9312 9313 9314, 9315, 9316, 9317, 9318 A4D2P2Rac1 UniProtKB: 9319 9320 9321, 9322, 9323, 9324, 9325 P63000 Rac1UniProtKB: 9326 9327 9328, 9329, 9330, 9331, 9332 WOUV93 RhoA UniProtKB:9333 9334 9335, 9336, 9337, 9338, 9339 A0A024R324 RhoA UniProtKB: 93409341 9342, 9343, 9344, 9345, 9346 P61586 RIG-I UniProtKB: 9347 93489349, 9350, 9351, 9352, 9353 O95786 RIG-I UniProtKB: 9354 9355 9356,9357, 9358, 9359, 9360 Q8IUD6 Src-Kinases UniProtKB: 9361 9362 9363,9364, 9365, 9366, 9367 Q9H5V8 Surfactant_protein_A UniProtKB: 9368 93699370, 9371, 9372, 9373, 9374 Q8IWL1 Surfactant_protein_A UniProtKB: 93759376 9377, 9378, 9379, 9380, 9381 Q8IWL2 Surfactant_protein_D UniProtKB:9382 9383 9384, 9385, 9386, 9387, 9388 P35247 TAK1 UniProtKB: 9389 93909391, 9392, 9393, 9394, 9395 O43318 TAK1 UniProtKB: 9396 9397 9398,9399, 9400, 9401, 9402 P49116 TBK1 UniProtKB: 9403 9404 9405, 9406,9407, 9408, 9409 Q9UHD2 TICAM1 UniProtKB: 9410 9411 9412, 9413, 9414,9415, 9416 Q8IUC6 TICAM2 UniProtKB: 9417 9418 9419, 9420, 9421, 9422,9423 Q86XR7 TIRAP UniProtKB: 9424 9425 9426, 9427, 9428, 9429, 9430A0A024R3M4 TIRAP UniProtKB: 9431 9432 9433, 9434, 9435, 9436, 9437P58753 TLR10 UniProtKB: 9438 9439 9440, 9441, 9442, 9443, 9444A0A024R9W4 TLR10 UniProtKB: 9445 9446 9447, 9448, 9449, 9450, 9451D1CS19 TLR10 UniProtKB: 9452 9453 9454, 9455, 9456, 9457, 9458 D1CS20TLR10 UniProtKB: 9459 9460 9461, 9462, 9463, 9464, 9465 D1CS24 TLR10UniProtKB: 9466 9467 9468, 9469, 9470, 9471, 9472 D1CS26 TLR10UniProtKB: 9473 9474 9475, 9476, 9477, 9478, 9479 D1CS27 TLR10UniProtKB: 9480 9481 9482, 9483, 9484, 9485, 9486 D1CS28 TLR10UniProtKB: 9487 9488 9489, 9490, 9491, 9492, 9493 D1CS29 TLR10UniProtKB: 9494 9495 9496, 9497, 9498, 9499, 9500 D1CS30 TLR10UniProtKB: 9501 9502 9503, 9504, 9505, 9506, 9507 Q9BXR5 TLR1 UniProtKB:9508 9509 9510, 9511, 9512, 9513, 9514 D1CS34 TLR1 UniProtKB: 9515 95169517, 9518, 9519, 9520, 9521 D1CS35 TLR1 UniProtKB: 9522 9523 9524,9525, 9526, 9527, 9528 D1CS36 TLR1 UniProtKB: 9529 9530 9531, 9532,9533, 9534, 9535 D1CS38 TLR1 UniProtKB: 9536 9537 9538, 9539, 9540,9541, 9542 D1CS42 TLR1 UniProtKB: 9543 9544 9545, 9546, 9547, 9548, 9549D1CS43 TLR1 UniProtKB: 9550 9551 9552, 9553, 9554, 9555, 9556 D1CS44TLR1 UniProtKB: 9557 9558 9559, 9560, 9561, 9562, 9563 Q15399 TLR1UniProtKB: 9564 9565 9566, 9567, 9568, 9569, 9570 Q5FWG5 TLR1 UniProtKB:9571 9572 9573, 9574, 9575, 9576, 9577 Q6F164 TLR2 UniProtKB: 9578 95799580, 9581, 9582, 9583, 9584 O60603 TLR3 UniProtKB: 9585 9586 9587,9588, 9589, 9590, 9591 O15455 TLR4 UniProtKB: 9592 9593 9594, 9595,9596, 9597, 9598 D1CS55 TLR4 UniProtKB: 9599 9600 9601, 9602, 9603,9604, 9605 O00206 TLR5 UniProtKB: 9606 9607 9608, 9609, 9610, 9611, 9612D1CS79 TLR5 UniProtKB: 9613 9614 9615, 9616, 9617, 9618, 9619 D1CS82TLR5 UniProtKB: 9620 9621 9622, 9623, 9624, 9625, 9626 D1CS83 TLR5UniProtKB: 9627 9628 9629, 9630, 9631, 9632, 9633 D1CS84 TLR5 UniProtKB:9634 9635 9636, 9637, 9638, 9639, 9640 D1CS85 TLR5 UniProtKB: 9641 96429643, 9644, 9645, 9646, 9647 D1CS87 TLR5 UniProtKB: 9648 9649 9650,9651, 9652, 9653, 9654 D1CS88 TLR5 UniProtKB: 9655 9656 9657, 9658,9659, 9660, 9661 D1CS89 TLR5 UniProtKB: 9662 9663 9664, 9665, 9666,9667, 9668 D1CS90 TLR6 UniProtKB: 9669 9670 9671, 9672, 9673, 9674, 9675B6CH37 TLR6 UniProtKB: 9676 9677 9678, 9679, 9680, 9681, 9682 B6CH42TLR6 UniProtKB: 9683 9684 9685, 9686, 9687, 9688, 9689 B6CH44 TLR6UniProtKB: 9690 9691 9692, 9693, 9694, 9695, 9696 B6CH45 TLR6 UniProtKB:9697 9698 9699, 9700, 9701, 9702, 9703 B6RFS7 TLR6 UniProtKB: 9704 97059706, 9707, 9708, 9709, 9710 D1CS91 TLR6 UniProtKB: 9711 9712 9713,9714, 9715, 9716, 9717 D1CS92 TLR6 UniProtKB: 9718 9719 9720, 9721,9722, 9723, 9724 D1CS93 TLR6 UniProtKB: 9725 9726 9727, 9728, 9729,9730, 9731 D1CS96 TLR6 UniProtKB: 9732 9733 9734, 9735, 9736, 9737, 9738D1CS97 TLR6 UniProtKB: 9739 9740 9741, 9742, 9743, 9744, 9745 D1CS98TLR6 UniProtKB: 9746 9747 9748, 9749, 9750, 9751, 9752 D1CS99 TLR6UniProtKB: 9753 9754 9755, 9756, 9757, 9758, 9759 D1CSA0 TLR7 UniProtKB:9760 9761 9762, 9763, 9764, 9765, 9766 B2R9N9 TLR7 UniProtKB: 9767 97689769, 9770, 9771, 9772, 9773 D1CS68 TLR7 UniProtKB: 9774 9775 9776,9777, 9778, 9779, 9780 Q9NYK1 TLR8 UniProtKB: 9781 9782 9783, 9784,9785, 9786, 9787 Q495P6 TLR8 UniProtKB: 9788 9789 9790, 9791, 9792,9793, 9794 Q495P7 TLR8 UniProtKB: 9795 9796 9797, 9798, 9799, 9800, 9801Q9NR97 TLR9 UniProtKB: 9802 9803 9804, 9805, 9806, 9807, 9808 B6CH46TLR9 UniProtKB: 9809 9810 9811, 9812, 9813, 9814, 9815 D1CS61 TLR9UniProtKB: 9816 9817 9818, 9819, 9820, 9821, 9822 D1CS62 TLR9 UniProtKB:9823 9824 9825, 9826, 9827, 9828, 9829 L0R5D6 TLR9 UniProtKB: 9830 98319832, 9833, 9834, 9835, 9836 L8E8B9 TLR9 UniProtKB: 9837 9838 9839,9840, 9841, 9842, 9843 Q9NR96 TRAF6 UniProtKB: 9844 9845 9846, 9847,9848, 9849, 9850 Q9Y4K3 c-myc UniProtKB: 9851 9852 9853, 9854, 9855,9856, 9857, A0A0B4J1R1 9858 c-myc UniProtKB: 9859 9860 9861, 9862, 9863,9864, 9865, P01106 9866 c-myc UniProtKB: 9867 9868 9869, 9870, 9871,9872, 9873, Q14901 9874 c-myc UniProtKB: 9875 9876 9877, 9878, 9879,9880, 9881, Q16591 9882

According to the present invention, in a more preferred embodiment, theinventive composition comprises at least one RNA, preferably an mRNAcomprising at least one coding region encoding at least one innateimmune activator or a fragment or variant thereof, wherein the at leastone coding region comprises an RNA sequence being identical or at least50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the RNAsequences according to the SEQ ID Nos as disclosed in Table 11.

13. Antibodies, Decoy Receptors and Dominant Negative Receptors:

According to a preferred embodiment the at least one RNA of theinventive RNA containing composition encodes at least one antibodyand/or at least one dominant negative receptor and/or at least one decoyreceptor or a fragment or variant thereof, modulating (e.g. inhibiting)the functionality of a protein or signaling pathway which is associatedwith tumor or cancer development. It is particularly preferred that theRNA containing composition comprises at least one RNA encoding the heavychain of a particular antibody or fragments or variants thereof and atleast one further RNA encoding the light chain of the same particularantibody or fragments or variants thereof.

In this context particularly preferred are the antibodies according toTable 12.

TABLE 12 Antibodies directed against proteins accociated with tumor orcancer development Name Target 3F8 GD2 Abagovomab CA-125 imitationAbciximab Platelet glycoprotein GPIIb/IIIa Adecatumumab EpCAM (CD326)Afutuzumab CD20 Alacizumab pegol VEGFR2 Alemtuzumab CD52 Altumomabpentetate CEA Amatuximab mesothelin Anatumomab mafenatox 5T4 Anetumabravtansine mesothelin Apolizumab HLA-DR beta apomab TRAIL-R2 (CD262)Arcitumomab CEA Ascrinvacumab ACVRL1 Bavituximab phosphatidylserineBectumomab CD22 Belimumab BAFF Besilesomab CEA Bevacizumab VEGF-ABivatuzumab mertansine CD44v6 Blinatumomab CD19 × CD3 Brentuximabvedotin CD30 (TNFRSF8) Brontictuzumab NOTCH1 canakinumab IL-1βCantuzumab mertansine CanAg Cantuzumab ravtansine MUC1 (CD227) Capromabpendetide PSMA Carlumab MCP-1 Catumaxomab EpCAM × CD3 cBR-doxorubicinimmunoconjugate CD174 (Lewis Y) Cetuximab EGFR (HER1/ERBB1) Citatuzumabbogatox EpCAM Cixutumumab IGF-1R Clivatuzumab tetraxetan MUC1 (CD227)Codrituzumab glypican 3 Coltuximab ravtansine CD19 Conatumumab TRAIL-R2(CD262) Dacetuzumab CD40 Dalotuzumab IGF-1R Dalotuzumab insulin-likegrowth factor I receptor Daratumumab CD38 (cyclic ADP ribose hydrolase)Demcizumab DLL4 Denintuzumab mafodotin CD19 Denosumab RANKLDepatuxizumab EGFR (HER1/ERBB1) Derlotuximab histone complex Detumomabunknown (B-lymphoma cells) Dinutuximab B4GALNT1 Drozitumab TRAIL-R2(CD262) Duligotumab HER3 (ERBB3) Duligotuzumab EGFR (HER1/ERBB1)Dusigitumab ILGF2 Ecromeximab GD3 ganglioside Edrecolomab EpCAMElgemtumab ERBB3 Elotuzumab SLAMF7 (CD319) Elsilimomab IL-6 EmactuzumabCSF1R Emibetuzumab HGFR Emibetuzumab MET Enavatuzumab TNFRSF12AEnfortumab vedotin AGS-22M6 Enoticumab DLL4 Ensituximab MUC5ACEpitumomab cituxetan MUC1 (CD227) Epratuzumab CD22 Ertumaxomab HER2(ERBB2/neu) × CD3 Etaracizumab integrin α5β3 Faralimomab IFNA1Farletuzumab FOLR1 alpha FBTA CD20 × CD3 Ficlatuzumab HGFR FigitumumabIGF-1R Flanvotumab TYRP1(glycoprotein 75) Fresolimumab TGF-β FutuximabEGFR (HER1/ERBB1) Galiximab CD80 Gantiumab IGF-1R Gemtuzumab ozogamicinCD33 Girentuximab Carbonic anhydrase 9 (CA9/CAIX) Glembatumumab vedotinGPNMB glycooptimized trastuzumab-GEX HER2 (ERBB2/neu) Ibritumomabtiuxetan CD20 Icrucumab VEGFR-1 Igovomab MUC16 IMAB362 Claudin-18(CLDN18.2) Imgatuzumab EGFR (HER1/ERBB1) Indatuximab ravtansine SDC1Indusatumab vedotin GUCY2C inebilizumab CD19 Inotuzumab ozogamicin CD22Intetumumab CD51 Iratumumab CD30 (TNFRSF8) Isatuximab CD38 LabetuzumabCEA Lenzilumab CSF2 Lexatumumab TRAIL-R2 (CD262) Lifastuzumab vedotinNaPi2B Lilotomab satetraxetan CD37 Lintuzumab CD33 Lorvotuzumabmertansine CD56 Lucatumumab CD40 Lumiliximab CD23 (IgE receptor)Lumretuzumab ERBB3 Mapatumumab TRAIL-R1 (CD261) Margetuximab HER2(ERBB2/neu) Matuzumab EGFR (HER1/ERBB1) Mepolizumab IL-5 MilatuzumabCD74 Minretumomab TAG-72 Mirvetuximab soravtansine FOLR1 alpha MitumomabGD3 (ganglioside) Mogamulizumab CCR4 Moxetumomab pasudotox CD22Nacolomab tafenatox C242 antigen Naptumomab estafenatox 5T4 NarnatumabRON Necitumumab EGFR (HER1/ERBB1) Nesvacumab ANGPT2 (angiopoietin 2)Nimotuzumab EGFR (HER1/ERBB1) Nofetumomab merpentan EpCAM binutuzumabCD20 Ocaratuzumab CD20 Ofatumumab CD20 Olaratumab PDGFRα Onartuzumab METOntuxizumab CD248 (TEM1) Oportuzumab monatox EpCAM Oregovomab CA-125Otlertuzumab CD37 Panitumumab EGFR (HER1/ERBB1) Pankomab MUC1 (tumorspecific glycosylation) Parsatuzumab EGFL7 Pasotuxizumab FOLH1Patritumab HER3 (ERBB3) Pemtumomab MUC1 (CD227) Pertuzumab HER2(ERBB2/neu) Pinatuzumab vedotin CD22 Pintumomab adenocarcinoma antigenPolatuzumab vedotin CD79B Racotumomab NGcGM3 Radretumab EDB (fibronectinextra domain-B) Ramucirumab VEGFR2 Rilotumumab HGFR Rituximab CD20Robatumumab IGF-1R Sacituzumab govitecan Trop-2 (tumor-associatedcalcium signal transducer 2/EGP-1) Samalizumab CD200 (OX-2 membraneglycoprotein) Satumomab pendetide TAG-72 Seribantumab ERBB3 SeribantumabHER3 (ERBB3) SGN-CDA CD19 SGN-CDA CD33 Sibrotuzumab FAP Siltuximab IL-6Simtuzumab LOXL2 Sofituzumab vedotin CA 125 Solitomab EpCAM SonepcizumabS1P (sphingosine-1-phosphate) Tacatuzumab tetraxetan AFP(alpha-fetoprotein) Taplitumomab paptox CD19 Tarextumab Notch receptorTenatumomab TN-C (tenascin C) Teprotumumab CD221 Tetulomab CD37 TGN CD28Tigatuzumab TRAIL-R2 (CD262) Lebrikizumab IL-13 Tocilizumab IL-6RTositumomab CD20 Tovetumab CD140a Tovetumab PDGFRα Trastuzumab HER2(ERBB2/neu) Trastuzumab emtansine HER2 (ERBB2/neu) TRBS GD2 Tucotuzumabcelmoleukin EpCAM ublituximab CD20 Ublituximab MS4A1 Ulocuplumab CXCR4Vandortuzumab vedotin STEAP1 Vantictumab FZD7 Vanucizumab Ang-2(angiopoietin 2) × VEGF-A Veltuzumab CD20 Vesencumab NRP1 Volociximabintegrin α5β1 Votumumab CTAA16.88 Zalutumumab EGFR (HER1/ERBB1)Zanolimumab CD4 Zatuximab HER1 (EGFR/ERBB1)

Preferably, the neutralizing antibody is chosen from the list ofanti-IL-10 and anti-TGFbeta.

Furthermore, the at least one antibody may preferably chosen fromanti-CD73 antibodies or fragments or variants thereof.

In a further particularly preferred embodiment the at least one antibodyis chosen from an antibody directed against CCR5/CD195 or from anantibody directed against its ligand CCL5/RANTES.

In a particularly preferred embodiment the decoy receptor is a solubleCCR5 (chemokine receptor type 5, also known as CD195).

In a further particularly preferred embodiment the dominant negativereceptor is dominant negative CCR5 (chemokine receptor type 5, alsoknown as CD195).

Furthermore, the at least one antibody may preferably chosen fromanti-CD73 antibodies or fragments or variants thereof.

14. Inhibitors of Myeloid Derived Suppressor Cells (MDSCs):

Myeloid Derived Suppressor Cells (MDSC) are a heterogeneous populationof immature myeloid cells that are increased in cancer and relateddisorders. MDSC are induced by tumor secreted growth factors. MDSC playan important part in suppression of host immune responses throughseveral mechanisms. In addition, MDSC may also contribute toangiogenesis and tumor invasion. Therefore, MDSC inhibition is astrategy for the treatment of cancer and related disorders.

In the context of the invention, MDSC inhibition can be achieved bydirect deactivation of MDSCs (e.g., anti IL-17 antibodies), by blockingdifferentiation of MDSCs into mature cells (e.g., IL-12), by blockingthe cell development of MDSCs or by depletion of MDSCs (e.g., cytotoxicagents).

Therefore it is particularly preferred to use anti IL-17 antibodies andIL-12 as inhibitors of MDSCs.

15. IDO Pathway Inhibitors

In a further preferred embodiment of the inventive RNA containingcomposition the RNA, preferably mRNA codes for at least one IDO pathwayinhibitor. Preferably the RNA encoding the at least one IDO pathwayinhibitor encodes an inhibitory protein or dominant negative mutantprotein of the IDO pathway.

As reviewed in Prendergast et al. (Prendergast G C, Smith C, Thomas S,Mandik-Nayak L, Laury-Kleintop L, Metz R, Muller A J. Indoleamine2,3-dioxygenase pathways of pathogenic inflammation and immune escape incancer. Cancer Immunol. Immunother. 2014 July; 63(7):721-35)indoleamine-pyrrole 2,3-dioxygenase (IDO or INDO EC 1.13.11.52) is anenzyme that in humans is encoded by the IDO1 gene. This enzyme catalyzesthe degradation of the essential amino acid L-tryptophan toN-formylkynurenine. IDO is the first and rate-limiting enzyme oftryptophan catabolism through kynurenine pathway, thus causing depletionof tryptophan which can cause halted growth of microbes as well as Tcells. IDO is an immunomodulatory enzyme produced by some alternativelyactivated macrophages and other immunoregulatory cells (also used as animmune subversion strategy by many tumors). The clinical development ofIDO inhibitors may produce a novel class of immunomodulators with broadapplication in the treatment of advanced human cancer.

16. Proteins or Peptides that Bind Inhibitors of Apoptosis

Apoptosis is a tightly regulated cellular process and faulty regulationof apoptosis is a hallmark of human cancers. Targeting key apoptosisregulators with the goal to restore apoptosis in tumor cells has beenpursued as a new cancer therapeutic strategy. XIAP, cIAP1, and cIAP2,members of inhibitor of apoptosis (IAP) proteins, are criticalregulators of cell death and survival and are attractive targets for newcancer therapy. The SMAC/DIABLO protein is an endogenous antagonist ofXIAP, cIAP1, and cIAP2. In the last decade, intense research effortshave resulted in the design and development of several small-moleculeSMAC mimetics now in clinical trials for cancer treatment

In a further preferred embodiment, the inventive composition comprisesat least one RNA comprising at least one coding region that codes for atleast one peptide or protein that binds inhibitors of apoptosis proteins(IAPB) and thus sensitize cancer cells to apoptotic death.

Therefore it is particularly preferred that the at least one RNA of theinventive RNA containing composition encodes at least one protein orpeptide that bind inhibitors of apoptosis, such as SMAC mimetics.

Particularly preferred proteins or peptides that bind IAPB according tothe present invention comprise Omi/HtrA2, Smac, Smac derived peptides,Smac/DIABLO, and XAF1 (XIAP-associated factor 1) and fragments orvariants thereof.

RNA Modifications

According to one embodiment, the at least one RNA of the composition,encoding at least one of the proteins and/or peptides defined herein,may be in the form of a modified RNA, wherein any modification, asdefined herein, may be introduced into the at least one RNA of thecomposition. Modifications as defined herein preferably lead to astabilization of the at least one RNA of the composition of the presentinvention.

According to one embodiment, the at least one RNA of the composition ofthe present invention may thus be provided as a “stabilized RNA”, thatis to say as an RNA that is essentially resistant to in vivo degradation(e.g. by an exo- or endo-nuclease). Such stabilization can be effected,for example, by a modified phosphate backbone of the at least one RNA ofthe composition of the present invention. A backbone modification inconnection with the present invention is a modification in whichphosphates of the backbone of the nucleotides contained in the RNA arechemically modified. Nucleotides that may be preferably used in thisconnection contain e.g. a phosphorothioate-modified phosphate backbone,preferably at least one of the phosphate oxygens contained in thephosphate backbone being replaced by a sulfur atom. Stabilized RNAs mayfurther include, for example: non-ionic phosphate analogues, such as,for example, alkyl and aryl phosphonates, in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group, orphosphodiesters and alkylphosphotriesters, in which the charged oxygenresidue is present in alkylated form. Such backbone modificationstypically include, without implying any limitation, modifications fromthe group consisting of methylphosphonates, phosphoramidates andphosphorothioates (e.g. cytidine-5′-O-(1-thiophosphate)).

In the following, specific modifications are described, which arepreferably capable of “stabilizing” the at least one RNA as definedherein.

Chemical Modifications:

The term “RNA modification” as used herein may refer to chemicalmodifications comprising backbone modifications as well as sugarmodifications or base modifications.

In this context, a modified RNA as defined herein may contain nucleotideanalogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in an RNA asdefined herein are chemically modified. A sugar modification inconnection with the present invention is a chemical modification of thesugar of the nucleotides of the RNA as defined herein. Furthermore, abase modification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the RNA. In thiscontext, nucleotide analogues or modifications are preferably selectedfrom nucleotide analogues, which are applicable for transcription and/ortranslation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified RNA as described herein, can be modified in the sugar moiety.For example, the 2′ hydroxyl group (OH) can be modified or replaced witha number of different “oxy” or “deoxy” substituents. Examples of“oxy”-2′ hydroxyl group modifications include, but are not limited to,alkoxy or aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar); polyethyleneglycols (PEG), —O(CH₂CH₂O)nCH₂CH₂OR;“locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected,e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar;and amino groups (—O-amino, wherein the amino group, e.g., NRR, can bealkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) oraminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into a modifiedRNA as described herein. The phosphate groups of the backbone can bemodified by replacing one or more of the oxygen atoms with a differentsubstituent. Further, the modified nucleosides and nucleotides caninclude the full replacement of an unmodified phosphate moiety with amodified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters.

Phosphorodithioates have both non-linking oxygens replaced by sulfur.The phosphate linker can also be modified by the replacement of alinking oxygen with nitrogen (bridged phosphoroamidates), sulfur(bridged phosphorothioates) and carbon (bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified RNA as described herein can further be modified in thenucleobase moiety. Examples of nucleobases found in RNA include, but arenot limited to, adenine, guanine, cytosine and uracil. For example, thenucleosides and nucleotides described herein can be chemically modifiedon the major groove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,I-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-I-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, I-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine,5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine or5′-O-(1-thiophosphate)-pseudouridine.

In further specific embodiments, a modified RNA may comprise nucleosidemodifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxyuridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine,7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine,N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine,N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine,7-deaza-adenosine.

Lipid Modification:

According to a further embodiment, a modified RNA as defined herein cancontain a lipid modification. Such a lipid-modified RNA typicallycomprises an RNA as defined herein. Such a lipid-modified RNA as definedherein typically further comprises at least one linker covalently linkedwith that RNA, and at least one lipid covalently linked with therespective linker. Alternatively, the lipid-modified RNA comprises atleast one RNA as defined herein and at least one (bifunctional) lipidcovalently linked (without a linker) with that RNA. According to a thirdalternative, the lipid-modified RNA comprises an RNA molecule as definedherein, at least one linker covalently linked with that RNA, and atleast one lipid covalently linked with the respective linker, and alsoat least one (bifunctional) lipid covalently linked (without a linker)with that RNA. In this context, it is particularly preferred that thelipid modification is present at the terminal ends of a linear RNAsequence.

G/C Content Optimization:

According to an especially preferred embodiment of the invention, theRNA of the inventive composition is modified. Preferably the RNA isstabilized by modifying and preferably increasing the G (guanosine)/C(cytosine) content of the RNA of the coding region thereof. Therein, theG/C content of the RNA of the coding region is increased compared to theG/C content of the coding region of its particular wild type codingsequence, i.e. the unmodified RNA. However, the encoded amino acidsequence of the RNA is preferably not modified compared to the encodedamino acid sequence of the particular wild type/unmodified RNA.

The modification of the G/C-content of the RNA of the inventivecomposition is based on the fact that RNA sequences having an increasedG (guanosine)/C (cytosine) content are more stable than RNA sequenceshaving an increased A (adenosine)/U (uracil) content. The codons of acoding sequence or a whole RNA might therefore be varied compared to thewild type coding sequence or RNA, such that they include an increasedamount of G/C nucleotides while the translated amino acid sequence isretained. In respect to the fact that several codons code for one andthe same amino acid (so-called degeneration of the genetic code), themost favourable codons for the stability can be determined (so-calledalternative codon usage). Depending on the amino acid to be encoded bythe at least one RNA, there are various possibilities for modificationof the RNA sequence, compared to its wild-type sequence. In the case ofamino acids which are encoded by codons, which contain exclusively G orC nucleotides, no modification of the codon is necessary. Thus, thecodons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly(GGC or GGG) require no modification, since no A or U is present. Incontrast, codons which contain A and/or U nucleotides can be modified bysubstitution of other codons, which code for the same amino acids butcontain no A and/or U. Examples of these are: the codons for Pro can bemodified from CCU or CCA to CCC or CCG; the codons for Arg can bemodified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Alacan be modified from GCU or GCA to GCC or GCG; the codons for Gly can bemodified from GGU or GGA to GGC or GGG. In other cases, although A or Unucleotides cannot be eliminated from the codons, it is however possibleto decrease the A and U content by using codons which contain a lowercontent of A and/or U nucleotides. Examples of these are: the codons forPhe can be modified from UUU to UUC; the codons for Leu can be modifiedfrom UUA, UUG, CUU or CUA to CUC or CUG; the codons for Ser can bemodified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyrcan be modified from UAU to UAC; the codon for Cys can be modified fromUGU to UGC; the codon for His can be modified from CAU to CAC; the codonfor Gln can be modified from CAA to CAG; the codons for Ile can bemodified from AUU or AUA to AUC; the codons for Thr can be modified fromACU or ACA to ACC or ACG; the codon for Asn can be modified from AAU toAAC; the codon for Lys can be modified from AAA to AAG; the codons forVal can be modified from GUU or GUA to GUC or GUG; the codon for Asp canbe modified from GAU to GAC; the codon for Glu can be modified from GAAto GAG; the stop codon UAA can be modified to UAG or UGA. In the case ofthe codons for Met (AUG) and Trp (UGG), on the other hand, there is nopossibility of sequence modification. The substitutions listed above canbe used either individually or in all possible combinations to increasethe G/C content of the at least one mRNA of the composition of thepresent invention compared to its particular wild-type mRNA (i.e. theoriginal sequence). Thus, for example, all codons for Thr occurring inthe wild-type sequence can be modified to ACC (or ACG). Preferably,however, for example, combinations of the above substitutionpossibilities are used:

substitution of all codons coding for Thr in the original sequence(wild-type mRNA) to ACC (or ACG) and substitution of all codonsoriginally coding for Ser to UCC (or UCG or AGC); substitution of allcodons coding for Ile in the original sequence to AUC and

substitution of all codons originally coding for Lys to AAG and

substitution of all codons originally coding for Tyr to UAC;substitution of all codons coding for Val in the original sequence toGUC (or GUG) and

substitution of all codons originally coding for Glu to GAG and

substitution of all codons originally coding for Ala to GCC (or GCG) and

substitution of all codons originally coding for Arg to CGC (or CGG);substitution of all codons coding for Val in the original sequence toGUC (or GUG) and

substitution of all codons originally coding for Glu to GAG and

substitution of all codons originally coding for Ala to GCC (or GCG) and

substitution of all codons originally coding for Gly to GGC (or GGG) and

substitution of all codons originally coding for Asn to AAC;substitution of all codons coding for Val in the original sequence toGUC (or GUG) and

substitution of all codons originally coding for Phe to UUC and

substitution of all codons originally coding for Cys to UGC and

substitution of all codons originally coding for Leu to CUG (or CUC) and

substitution of all codons originally coding for Gln to CAG and

substitution of all codons originally coding for Pro to CCC (or CCG);etc.

Preferably, the G/C content of the coding region of the at least one RNAaccording to the invention is increased by at least 7%, more preferablyby at least 15%, particularly preferably by at least 20%, compared tothe G/C content of the coding region of the wild type RNA. According toa specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, morepreferably at least 70%, even more preferably at least 80% and mostpreferably at least 90%, 95% or even 100% of the substitutable codons inthe region coding for a protein or peptide as defined herein or itsfragment or variant thereof or the whole sequence of the wild type RNAsequence or coding sequence are substituted, thereby increasing the G/Ccontent of said sequence. In this context, it is particularly preferableto increase the G/C content of the at least one RNA of the inventivecomposition to the maximum (i.e. 100% of the substitutable codons), inparticular in the coding region, compared to the wild type sequence.

According to the invention, a further preferred modification of thecoding sequence of the at least one RNA of the composition is based onthe finding that the translation efficiency is also determined by adifferent frequency in the occurrence of tRNAs in cells. Thus, ifso-called “rare codons” are present in the at least one coding region ofthe at least one RNA of the composition of the present invention to anincreased extent, the corresponding modified at least one RNA sequenceis translated to a significantly poorer degree than in the case wherecodons coding for relatively “frequent” tRNAs are present. According tothe invention, in the modified at least one RNA of the composition ofthe present invention, the region which codes for one of the abovedefined peptides or proteins is modified compared to the correspondingregion of the wild-type RNA such that at least one codon of thewild-type sequence, which codes for a tRNA which is relatively rare inthe cell, is exchanged for a codon, which codes for a tRNA which isrelatively frequent in the cell and carries the same amino acid as therelatively rare tRNA. By this modification, the sequence of the at leastone coding region of the at least one RNA of the composition of thepresent invention is modified such that codons for which frequentlyoccurring tRNAs are available are inserted. In other words, according tothe invention, by this modification all codons of the wild-type sequencewhich code for a tRNA which is relatively rare in the cell can in eachcase be exchanged for a codon which codes for a tRNA which is relativelyfrequent in the cell and which, in each case, carries the same aminoacid as the relatively rare tRNA. Which tRNAs occur relativelyfrequently in the cell and which, in contrast, occur relatively rarelyis known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin.Genet. Dev. 2001, 11(6): 660-666. The codons which use for theparticular amino acid the tRNA which occurs the most frequently, e.g.the Gly codon, which uses the tRNA, which occurs the most frequently inthe (human) cell, are particularly preferred. According to theinvention, it is particularly preferable to link the sequential G/Ccontent which is increased, in particular maximized, in the modified atleast one RNA of the composition of the present invention, with the“frequent” codons without modifying the amino acid sequence of theprotein encoded by the coding region of the RNA. This preferredembodiment allows provision of a particularly efficiently translated andstabilized (modified) at least one RNA of the composition of the presentinvention. The determination of a modified at least one RNA of thecomposition of the present invention as described above (increased G/Ccontent; exchange of tRNAs) can be carried out using the computerprogram explained in WO 02/098443—the disclosure content of which isincluded in its full scope in the present invention. Using this computerprogram, the nucleotide sequence of any desired coding RNA can bemodified with the aid of the genetic code or the degenerative naturethereof such that a maximum G/C content results, in combination with theuse of codons which code for tRNAs occurring as frequently as possiblein the cell, the amino acid sequence coded by the modified at least oneRNA preferably not being modified compared to the non-modified sequence.Alternatively, it is also possible to modify only the G/C content oronly the codon usage compared to the original sequence. The source codein Visual Basic 6.0 (development environment used: Microsoft VisualStudio Enterprise 6.0 with Servicepack 3) is also described in WO02/098443. In a further preferred embodiment of the present invention,the A/U content in the environment of the ribosome binding site of theat least one RNA of the composition of the present invention isincreased compared to the A/U content in the environment of the ribosomebinding site of its particular wild-type RNA. This modification (anincreased A/U content around the ribosome binding site) increases theefficiency of ribosome binding to the at least one RNA. An effectivebinding of the ribosomes to the ribosome binding site (Kozak sequence:GCCGCCACCAUGG (SEQ ID NO: 10.071), the AUG forms the start codon) inturn has the effect of an efficient translation of the at least one RNA.According to a further embodiment of the present invention the at leastone RNA of the composition of the present invention may be modified withrespect to potentially destabilizing sequence elements. Particularly,the coding region and/or the 5′ and/or 3′ untranslated region of thisRNA may be modified compared to the particular wild-type RNA such thatit contains no destabilizing sequence elements, the coded amino acidsequence of the modified at least one RNA preferably not being modifiedcompared to its particular wild-type RNA. It is known that, for example,in sequences of eukaryotic RNAs destabilizing sequence elements (DSE)occur, to which signal proteins bind and regulate enzymatic degradationof RNA in vivo. For further stabilization of the modified at least oneRNA, optionally in the region which encodes for a protein or peptide asdefined herein, one or more such modifications compared to thecorresponding region of the wild-type RNA can therefore be carried out,so that no or substantially no destabilizing sequence elements arecontained there. According to the invention, DSE present in theuntranslated regions (3′- and/or 5′-UTR) can also be eliminated from theat least one RNA of the composition of the present invention by suchmodifications. Such destabilizing sequences are e.g. AU-rich sequences(AURES), which occur in 3′-UTR sections of numerous unstable RNAs (Caputet al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). The at leastone RNA of the composition of the present invention is thereforepreferably modified compared to the wild-type RNA such that the at leastone RNA contains no such destabilizing sequences. This also applies tothose sequence motifs which are recognized by possible endonucleases,e.g. the sequence GAACAAG, which is contained in the 3′-UTR segment ofthe gene which codes for the transferrin receptor (Binder et al., EMBOJ. 1994, 13: 1969 to 1980). These sequence motifs are also preferablyremoved in the at least one RNA of the composition of the presentinvention.

Adaptation to Human Codon Usage:

According to the invention, a further preferred modification of the atleast one RNA of the composition of the present invention is based onthe finding that codons coding for the same amino acid occur indifferent frequencies. According to the invention, in the modified atleast one RNA of the composition of the present invention, the regionwhich codes for one of the above defined peptides or proteins (codingsequence) is preferably modified compared to the corresponding region ofthe wild-type RNA such that the frequency of the codons coding for thesame amino acid corresponds to the naturally occurring frequency of thatcodon present in the human coding usage as e.g. shown in Table 13.

This means, for example, that for the amino acid Alanine (Ala) presentin the amino acid sequence of the encoded protein according to theinvention, the wild type coding sequence is adapted in a way that thecodon “GCC” is used with a frequency of 0.40, the codon “GCT” is usedwith a frequency of 0.28, the codon “GCA” is used with a frequency of0.22 and the codon “GCG” is used with a frequency of 0.10 etc. (seeTable 13).

TABLE 13 Human codon usage table (most frequent codonmarked with an asterisk) Amino acid codon fraction /1000 Ala GCG 0.107.4 Ala GCA 0.22 15.8 Ala GCT 0.28 18.5 Ala  GCC* 0.40 27.7 Cys TGT 0.4210.6 Cys  TGC* 0.58 12.6 Asp GAT 0.44 21.8 Asp  GAC* 0.56 25.1 Glu  GAG*0.59 39.6 Glu GAA 0.41 29.0 Phe TTT 0.43 17.6 Phe  TTC* 0.57 20.3 GlyGGG 0.23 16.5 Gly GGA 0.26 16.5 Gly GGT 0.18 10.8 Gly  GGC* 0.33 22.2His CAT 0.41 10.9 His  CAC* 0.59 15.1 Ile ATA 0.14 7.5 Ile ATT 0.35 16.0Ile  ATC* 0.52 20.8 Lys  AAG* 0.60 31.9 Lys AAA 0.40 24.4 Leu TTG 0.1212.9 Leu TTA 0.06 7.7 Leu  CTG* 0.43 39.6 Leu CTA 0.07 7.2 Leu CTT 0.1213.2 Leu CTC 0.20 19.6 Met  ATG* 1 22.0 Asn AAT 0.44 17.0 Asn  AAC* 0.5619.1 Pro CCG 0.11 6.9 Pro CCA 0.27 16.9 Pro CCT 0.29 17.5 Pro  CCC* 0.3319.8 Gln  CAG* 0.73 34.2 Gln CAA 0.27 12.3 Arg AGG 0.22 12.0 Arg  AGA*0.21 12.1 Arg CGG 0.19 11.4 Arg CGA 0.10 6.2 Arg CGT 0.09 4.5 Arg CGC0.19 10.4 Ser AGT 0.14 12.1 Ser  AGC* 0.25 19.5 Ser TCG 0.06 4.4 Ser TCA0.15 12.2 Ser TCT 0.18 15.2 Ser TCC 0.23 17.7 Thr ACG 0.12 6.1 Thr ACA0.27 15.1 Thr ACT 0.23 13.1 Thr  ACC* 0.38 18.9 Val  GTG* 0.48 28.1 ValGTA 0.10 7.1 Val GTT 0.17 11.0 Val GTC 0.25 14.5 Trp  TGG* 1 13.2 TyrTAT 0.42 12.2 Tyr  TAC* 0.58 15.3 Stop  TGA* 0.61 1.6 Stop TAG 0.17 0.8Stop TAA 0.22 1.0

Codon-Optimization:

According to a particularly preferred embodiment it is preferred, thatall codons of the wild-type sequence of the coding region of the atleast one RNA of the inventive composition which code for a tRNA whichis relatively rare in the cell is in each case exchanged for a codonwhich codes for a tRNA which is relatively frequent in the cell andwhich, in each case, carries the same amino acid as the relatively raretRNA. Therefore it is particularly preferred that the most frequentcodons are used for each encoded amino acid (see Table 13, most frequentcodons are marked with asterisks).

This means, for example, that for the amino acid Alanine (Ala) presentin the amino acid sequence of the encoded peptide or protein accordingto the invention, the wild type coding sequence is adapted in a way thatthe most frequent human codon “GCC” is always used for said amino acid,or for the amino acid Cysteine (Cys), the wild type sequence is adaptedin a way that the most frequent human codon “TGC” is always used forsaid amino acid etc.

C-Enrichment:

According to another embodiment, the at least one RNA of the compositionof the present invention may be modified by increasing the C content ofthe RNA, preferably of the coding region of the at least one RNA.

In a particularly preferred embodiment of the present invention, the Ccontent of the coding region of the at least one RNA of the compositionof the present invention is modified, particularly increased, comparedto the C content of the coding region of its particular wild-type RNA,i.e. the unmodified mRNA. The amino acid sequence encoded by the atleast one RNA is preferably not modified as compared to the amino acidsequence encoded by the particular wild-type RNA

In a preferred embodiment of the present invention, the modified RNA ismodified such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, orat least 90% of the theoretically maximal cytosine-content or even amaximal cytosine-content is achieved.

In further preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or even 100% of the codons of the target RNA wild typesequence, which are “cytosine content optimizable” are replaced bycodons with a higher cytosine-content as present in the wild typesequence.

In a further preferred embodiment, some of the codons of the wild typecoding sequence may additionally be modified such that a codon for arelatively rare tRNA in the cell is exchanged by a codon for arelatively frequent tRNA in the cell, provided that the substitutedcodon for a relatively frequent tRNA carries the same amino acid as therelatively rare tRNA of the original wild type codon. Preferably, all ofthe codons for a relatively rare tRNA are replaced by a codon for arelatively frequent tRNA in the cell, except codons encoding aminoacids, which are exclusively encoded by codons not containing anycytosine, or except for glutamine (Gln), which is encoded by two codonseach containing the same number of cytosines.

In a further preferred embodiment of the present invention, the modifiedtarget RNA is modified such that at least 80%, or at least 90% of thetheoretically maximal cytosine-content or even a maximalcytosine-content is achieved by means of codons, which code forrelatively frequent tRNAs in the cell, wherein the amino acid sequenceremains unchanged.

Due to the naturally occurring degeneracy of the genetic code, more thanone codon may encode a particular amino acid. Accordingly, 18 out of 20naturally occurring amino acids are encoded by more than 1 codon (withTryp and Met being an exception), e.g. by 2 codons (e.g. Cys, Asp, Glu),by three codons (e.g. Ile), by 4 codons (e.g. Al, Gly, Pro) or by 6codons (e.g. Leu, Arg, Ser). However, not all codons encoding the sameamino acid are utilized equally frequent under in vivo conditions.Depending on each single organism, a typical codon usage profile isestablished.

The term “cytosine content-optimizable codon” as used within the contextof the present invention refers to codons, which exhibit a lower amountof cytosines than other codons coding for the same amino acid.Accordingly, any wild type codon, which may be replaced by another codoncoding for the same amino acid and exhibiting a higher number ofcytosines within that codon, is considered to be cytosine-optimizable(C-optimizable). Any such substitution of a C-optimizable wild typecodon by the specific C-optimized codon within a wild type coding regionincreases its overall C-content and reflects a C-enriched modified RNAsequence. A C-maximized RNA sequence contains C-optimized codons for allpotentially C-optimizable codons. Accordingly, 100% or all of thetheoretically replaceable C-optimizable codons are under such conditionsactually replaced by C-optimized codons over the entire length of thecoding region.

In this context, cytosine-content optimizable codons are codons, whichcontain a lower number of cytosines than other codons coding for thesame amino acid.

Any of the codons GCG, GCA, GCU codes for the amino acid Ala, which maybe exchanged by the codon GCC encoding the same amino acid, and/or

the codon UGU that codes for Cys may be exchanged by the codon UGCencoding the same amino acid, and/or

the codon GAU which codes for Asp may be exchanged by the codon GACencoding the same amino acid, and/or

the codon that UUU that codes for Phe may be exchanged for the codon UUCencoding the same amino acid, and/or

any of the codons GGG, GGA, GGU that code Gly may be exchanged by thecodon GGC encoding the same amino acid, and/or

the codon CAU that codes for His may be exchanged by the codon CACencoding the same amino acid, and/or

any of the codons AUA, AUU that code for Ile may be exchanged by thecodon AUC, and/or

any of the codons UUG, UUA, CUG, CUA, CUU coding for Leu may beexchanged by the codon CUC encoding the same amino acid, and/or

the codon AAU that codes for Asn may be exchanged by the codon AACencoding the same amino acid, and/or

any of the codons CCG, CCA, CCU coding for Pro may be exchanged by thecodon CCC encoding the same amino acid, and/or

any of the codons AGG, AGA, CGG, CGA, CGU coding for Arg may beexchanged by the codon CGC encoding the same amino acid, and/or

any of the codons AGU, AGC, UCG, UCA, UCU coding for Ser may beexchanged by the codon UCC encoding the same amino acid, and/or

any of the codons ACG, ACA, ACU coding for Thr may be exchanged by thecodon ACC encoding the same amino acid, and/or

any of the codons GUG, GUA, GUU coding for Val may be exchanged by thecodon GUC encoding the same amino acid, and/or

the codon UAU coding for Tyr may be exchanged by the codon UAC encodingthe same amino acid.

In any of the above instances, the number of cytosines is increased by 1per exchanged codon. Exchange of all non C-optimized codons(corresponding to C-optimizable codons) of the coding region results ina C-maximized coding sequence. In the context of the invention at least70% of the non C-optimized codons are replaced by C-optimized codons ofthe wild type sequence are replaced by C-optimized codons, preferably atleast 80%, more preferably at least 90% within the coding region. It maybe preferred that for some amino acids the percentage of C-optimizablecodons replaced by C-optimized codons is less than 70%, while for otheramino acids the percentage of replaced codons is higher than 70% to meetthe overall percentage of C-optimization of at least 70% of allC-optimizable wild type codons of the coding region.

Preferably, in the C-optimized RNAs of the invention, at least 50% ofthe C-optimizable wild type codons for any given amino acid are replacedby C-optimized codons, e.g. any modified C-enriched RNA preferablycontains at least 50% C-optimized codons at C-optimizable wild typecodon positions coding for any single of the above mentioned amino acidsAla, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val andTyr, preferably at least 60%.

In this context codons coding for amino acids, which are not cytosinecontent-optimizable and which are, however, encoded by at least twocodons, may be used without any further selection process. However, thecodon of the wild type sequence that codes for a relatively rare tRNA inthe cell, e.g. a human cell, may be exchanged for a codon that codes fora relatively frequent tRNA in the cell, whereby both code for the sameamino acid. Accordingly, the relatively rare codon GAA coding for Glumay be exchanged by the relative frequent codon GAG coding for the sameamino acid, and/or

the relatively rare codon AAA coding for Lys may be exchanged by therelative frequent codon AAG coding for the same amino acid, and/or

the relatively rare codon CAA coding for Gln is exchanged for therelative frequent codon CAG encoding the same amino acid.

In this context, the amino acids Met (AUG) and Trp (UGG), which areencoded by only one codon each, remain unchanged. Stop codons are notcytosine-content optimized, however, the relatively rare stop codonsamber, ochre (UAA, UAG) may be exchanged by the relatively frequent stopcodon opal (UGA).

The substitutions listed above may obviously be used individually butalso in all possible combinations in order to optimize thecytosine-content of the modified RNA compared to the wild type RNAsequence.

Accordingly, the region of the modified RNA coding for the peptide orprotein may be changed compared to the coding region of the wild typeRNA in such a way that an amino acid encoded by at least two or morecodons, of which one comprises one additional cytosine, such a codon maybe exchanged by the C-optimized codon comprising one additionalcytosine, whereby the amino acid is unaltered compared to the wild typesequence.

Substitutions, additions or eliminations of bases are preferably carriedout using a DNA matrix for preparation of the nucleic acid molecule bytechniques of the well known site directed mutagenesis or with anoligonucleotide ligation. In such a process, for preparation of the atleast one RNA as defined herein a corresponding DNA molecule may betranscribed in vitro. This DNA matrix preferably comprises a suitablepromoter, e.g. a T7 or SP6 promoter, for in vitro transcription, whichis followed by the desired nucleotide sequence for the at least one RNAto be prepared and a termination signal for in vitro transcription. TheDNA molecule, which forms the matrix of the at least one RNA ofinterest, may be prepared by fermentative proliferation and subsequentisolation as part of a plasmid which can be replicated in bacteria.Plasmids which may be mentioned as suitable for the present inventionare e.g. the plasmids pT7Ts (GenBank accession number U26404; Lai etal., Development 1995, 121: 2349 to 2360), pGEM® series, e.g. pGEM®-1(GenBank accession number X65300; from Promega) and pSP64 (GenBankaccession number X65327); cf. also Mezei and Storts, Purification of PCRProducts, in: Griffin and Griffin (ed.), PCR Technology: CurrentInnovation, CRC Press, Boca Raton, Fla., 2001.

Fragments and Variants

In the context of the invention additionally to the here disclosedpeptides and proteins, which show a certain degree of identity ofsequence, are incorporated. Therefore fragments and variants of theproteins and peptides as defined herein are disclosed herewith in thecontext of the present invention.

Furthermore fragments and variants of nucleic acids as defined hereinare therefore disclosed herewith in the context of the presentinvention.

Mono-Bi-Multicistronic, Self Cleaving Peptides Etc:

The coding region of the at least one RNA of the inventive compositionmay occur as a mono-, di-, or even multicistronic RNA, i.e. an RNAsequence which carries the coding sequences of one, two or more proteinsor peptides. Such coding sequences of the di-, or even multicistronicRNAs may be separated by at least one internal ribosome entry site(IRES) sequence. Thus, the at least one RNA according to the inventionmay further comprise one or more internal ribosome entry site (IRES)sequences or IRES-motifs, which may separate several open readingframes, especially if the RNA encodes for two or more peptides orproteins (bi- or multicistronic RNA). For example, the internal ribosomeentry site sequence may be derived from EMCV (encephalomyocarditisvirus) or from FMDV (Foot and mouth disease virus). Furthermoreself-cleaving signal peptides may be used which induce the cleavage ofthe resulting polypeptide which comprises several proteins or peptides,e.g. a self-cleaving signal peptide sequence derived from F2A peptidefrom FMDV.

Combinations of Different Coding Sequences

In a preferred embodiment, the inventive composition comprises at leastone, two, three, four, five, six, seven, eight, nine, ten or more RNAs,each comprising at least one, two, three, four, five, six, seven, eight,nine, ten or more coding regions encoding at least one or more cytokineas defined above and/or at least one or more chemokine as defined above,and/or at least one or more suicide gene product as defined above,and/or at least one or more immunogenic peptide or protein as definedabove, and/or at least one or more apoptosis inducer as defined above,and/or at least one or more angiogenesis inhibitor as defined above,and/or at least one or more heat shock protein as defined above, and/orat least one or more tumor antigen as defined above, and/or at least oneor more β-catenin inhibitor as defined above, and/or at least one ormore STING pathway activator as defined above, and/or at least one ormore checkpoint modulator as defined above, and/or at least one or moreinnate immune activator, and/or at least one or more antibody as definedabove, and/or at least one dominant negative receptor and/or at leastone or more decoy receptor, and/or at least one or more inhibitor ofmyeloid derived suppressor cells (MDSCs), and/or at least one or moreIDO pathway inhibitor, and/or at least one or more protein or peptidethat bind apoptosis inhibitors as defined above, or variants orfragments thereof.

Untranslated Regions (UTRs)

By a further embodiment the at least one RNA of the inventivecomposition preferably comprises at least one of the followingstructural elements: a 5′- and/or 3′-untranslated region element (UTRelement), particularly a 5′-UTR element which comprises or consists of anucleic acid sequence which is derived from the 5′-UTR of a TOP gene orfrom a fragment, homolog or a variant thereof, or a 5′- and/or 3′-UTRelement which may be derivable from a gene that provides a stable mRNAor from a homolog, fragment or variant thereof; a histone stem-loopstructure, preferably a histone stem-loop in its 3′ untranslated region;a 5′-CAP structure; a poly-A tail (poly(A) sequence); or a poly(C)sequence.

In a preferred embodiment the at least one RNA comprises at least one5′- or 3′-UTR element. In this context an UTR element comprises orconsists of a nucleic acid sequence which is derived from the 5′- or3′-UTR of any naturally occurring gene or which is derived from afragment, a homolog or a variant of the 5′- or 3′-UTR of a gene.Preferably the 5′- or 3′-UTR element used according to the presentinvention is heterologous to the coding region of the RNA of theinventive composition. Even if 5′- or 3′-UTR elements derived fromnaturally occurring genes are preferred, also synthetically engineeredUTR elements may be used in the context of the present invention.

In a particularly preferred embodiment the at least one RNA comprises atleast one 5′-untranslated region element (5′-UTR element) whichcomprises or consists of a nucleic acid sequence which is derived fromthe 5′-UTR of a TOP gene or which is derived from a fragment, homolog orvariant of the 5′-UTR of a TOP gene.

It is particularly preferred that the 5′-UTR element does not comprise aTOP-motif or a 5′-TOP, as defined above.

In some embodiments, the nucleic acid sequence of the 5′-UTR elementwhich is derived from a 5′-UTR of a TOP gene terminates at its 3′-endwith a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10upstream of the start codon (e.g. A(U/T)G) of the gene or mRNA it isderived from. Thus, the 5′-UTR element does not comprise any part of theprotein coding region. Thus, preferably, the only protein coding part ofmRNA of the inventive composition is provided by the coding region.

The nucleic acid sequence which is derived from the 5′-UTR of a TOP geneis preferably derived from a eukaryotic TOP gene, preferably a plant oranimal TOP gene, more preferably a chordate TOP gene, even morepreferably a vertebrate TOP gene, most preferably a mammalian TOP gene,such as a human TOP gene.

For example, the 5′-UTR element is preferably selected from 5′-UTRelements comprising or consisting of a nucleic acid sequence which isderived from a nucleic acid sequence selected from the group consistingof SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.1422 of the patent application WO2013/143700, whose disclosure isincorporated herein by reference, from the homologs of SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700, from a variant thereof, or preferablyfrom a corresponding RNA sequence. The term “homologs of SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700” refers to sequences of other speciesthan homo sapiens, which are homologous to the sequences according toSEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422of the patent application WO2013/143700.

In a preferred embodiment, the 5′-UTR element comprises or consists of anucleic acid sequence which is derived from a nucleic acid sequenceextending from nucleotide position 5 (i.e. the nucleotide that islocated at position 5 in the sequence) to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700 from a variant thereof, or a corresponding RNAsequence. It is particularly preferred that the 5′-UTR element isderived from a nucleic acid sequence extending from the nucleotideposition immediately 3′ to the 5′-TOP to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence.

In a particularly preferred embodiment, the 5′-UTR element comprises orconsists of a nucleic acid sequence which is derived from a 5′-UTR of aTOP gene encoding a ribosomal protein or from a variant of a 5′-UTR of aTOP gene encoding a ribosomal protein. For example, the 5′-UTR elementcomprises or consists of a nucleic acid sequence which is derived from a5′-UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67,170, 193, 244, 259, 554, 650, 675, 700, 721, 913, 1016, 1063, 1120,1138, and 1284-1360 of the patent application WO2013/143700, acorresponding RNA sequence, a homolog thereof, or a variant thereof asdescribed herein, preferably lacking the 5′-TOP motif. As describedabove, the sequence extending from position 5 to the nucleotideimmediately 5′ to the ATG (which is located at the 3′end of thesequences) corresponds to the 5′-UTR of said sequences.

Preferably, the 5′-UTR element comprises or consists of a nucleic acidsequence which is derived from a 5′-UTR of a TOP gene encoding aribosomal large protein (RPL) or from a homolog or variant of a 5′-UTRof a TOP gene encoding a ribosomal large protein (RPL). For example, the5′-UTR element comprises or consists of a nucleic acid sequence which isderived from a 5′-UTR of a nucleic acid sequence according to any of SEQID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and1422 of the patent application WO2013/143700, a corresponding RNAsequence, a homolog thereof, or a variant thereof as described herein,preferably lacking the 5′-TOP motif.

In a particularly preferred embodiment, the 5′-UTR element comprises orconsists of a nucleic acid sequence which is derived from the 5′-UTR ofa ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, or from a variant of the 5′-UTRof a ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, wherein preferably the 5′-UTRelement does not comprise the 5′-TOP of said gene.

A preferred sequence for a 5′-UTR element corresponds to SEQ ID No. 1368of the patent application WO2013/143700.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 20%, preferably of at least about 40%, preferably ofat least about 50%, preferably of at least about 60%, preferably of atleast about 70%, more preferably of at least about 80%, more preferablyof at least about 90%, even more preferably of at least about 95%, evenmore preferably of at least about 99% to the nucleic acid sequence asmentioned above (according to SEQ ID NO. 10.051 (5′-UTR of humanribosomal protein Large 32 lacking the 5′ terminal oligopyrimidinetract: GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC; corresponding to SEQID No. 1368 of the patent application WO2013/143700)) or preferably to acorresponding RNA sequence, or wherein the at least one 5′UTR elementcomprises or consists of a fragment of a nucleic acid sequence which hasan identity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID NO.10.052 or more preferably to a corresponding RNA sequence, wherein,preferably, the fragment is as described above, i.e. being a continuousstretch of nucleotides representing at least 20% etc. of the full-length5′-UTR.

Preferably, the fragment exhibits a length of at least about 20nucleotides or more, preferably of at least about 30 nucleotides ormore, more preferably of at least about 40 nucleotides or more.Preferably, the fragment is a functional fragment as described herein.

In some embodiments, the mRNA of the inventive composition comprises a5′-UTR element which comprises or consists of a nucleic acid sequencewhich is derived from the 5′-UTR of a vertebrate TOP gene, such as amammalian, e.g. a human TOP gene, selected from RPSA, RPS2, RPS3, RPS3A,RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14,RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24,RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5,RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13,RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23,RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32,RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40,RPL41, RPLP0, RPLP1, RPLP2, RPLP3, RPLP0, RPLP1, RPLP2, EEF1A1, EEF1B2,EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP,EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2,UQCRB, or from a homolog or variant thereof, wherein preferably the5′-UTR element does not comprise a TOP-motif or the 5′-TOP of saidgenes, and wherein optionally the 5′-UTR element starts at its 5′-endwith a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10downstream of the 5′ terminal oligopyrimidine tract (TOP) and whereinfurther optionally the 5′-UTR element which is derived from a 5′-UTR ofa TOP gene terminates at its 3′-end with a nucleotide located atposition 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon(A(U/T)G) of the gene it is derived from.

In further particularly preferred embodiments, the 5′-UTR elementcomprises or consists of a nucleic acid sequence which is derived fromthe 5′-UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomalprotein Large 35 gene (RPL35), a ribosomal protein Large 21 gene(RPL21), an ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, an hydroxysteroid(17-beta) dehydrogenase 4 gene (HSD17B4), an androgen-induced 1 gene(AIG1), cytochrome c oxidase subunit Vlc gene (COX6C), or aN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, preferably from a vertebrate ribosomal proteinLarge 32 gene (RPL32), a vertebrate ribosomal protein Large 35 gene(RPL35), a vertebrate ribosomal protein Large 21 gene (RPL21), avertebrate ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, a vertebratehydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a vertebrateandrogen-induced 1 gene (AIG1), a vertebrate cytochrome c oxidasesubunit Vlc gene (COX6C), or a vertebrate N-acylsphingosineamidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variantthereof, more preferably from a mammalian ribosomal protein Large 32gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomalprotein Large 21 gene (RPL21), a mammalian ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle(ATP5A1) gene, a mammalian hydroxysteroid (17-beta) dehydrogenase 4 gene(HSD17B4), a mammalian androgen-induced 1 gene (AIG1), a mammaliancyto-chrome c oxidase subunit Vlc gene (COX6C), or a mammalianN-acylsphingosine ami-dohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, most preferably from a human ribosomal proteinLarge 32 gene (RPL32), a human ribosomal protein Large 35 gene (RPL35),a human ribosomal protein Large 21 gene (RPL21), a human ATP synthase,H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiacmuscle (ATP5A1) gene, a human hydroxysteroid (17-beta) dehydrogenase 4gene (HSD17B4), a human androgen-induced 1 gene (AIG1), a humancytochrome c oxidase subunit Vlc gene (COX6C), or a humanN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, wherein preferably the 5′-UTR element does notcomprise the 5′-TOP of said gene.

In this context particularly preferred are 5′-UTR elements comprising anucleic acid sequence according to SEQ ID Nos. 10.051-10.054.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 1368, or SEQ IDNOs 1412-1420 of the patent application WO2013/143700, or acorresponding RNA sequence, or wherein the at least one 5′-UTR elementcomprises or consists of a fragment of a nucleic acid sequence which hasan identity of at least about 20%, preferably of at least about 40%,preferably of at least about 50%, preferably of at least about 60%,preferably of at least about 70%, more preferably of at least about 80%,more preferably of at least about 90%, even more preferably of at leastabout 95%, even more preferably of at least about 99% to the nucleicacid sequence according to SEQ ID No. 1368, or SEQ ID NOs 1412-1420 ofthe patent application WO2013/143700, wherein, preferably, the fragmentis as described above, i.e. being a continuous stretch of nucleotidesrepresenting at least 20% etc. of the full-length 5′-UTR. Preferably,the fragment exhibits a length of at least about 20 nucleotides or more,preferably of at least about 30 nucleotides or more, more preferably ofat least about 40 nucleotides or more. Preferably, the fragment is afunctional fragment as described herein.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 20%, preferably of at least about 40%, preferably ofat least about 50%, preferably of at least about 60%, preferably of atleast about 70%, more preferably of at least about 80%, more preferablyof at least about 90%, even more preferably of at least about 95%, evenmore preferably of at least about 99% to the nucleic acid sequenceaccording to SEQ ID No. 10.053 (5′-UTR of ATP5A1 lacking the 5′ terminaloligopyrimidine tract:GCGGCTCGGCCATTTTGTCCCAGTCAGTCCGGAGGCTGCGGCTGCAGAAGTACCGCCTGCG-GAGTAACTGCAAAG;corresponding to SEQ ID No. 1414 of the patent application WO2013/143700(5′-UTR of ATP5A1 lacking the 5′ terminal oligopyrimidine tract) orpreferably to a corresponding RNA sequence, or wherein the at least one5′UTR element comprises or consists of a fragment of a nucleic acidsequence which has an identity of at least about 40%, preferably of atleast about 50%, preferably of at least about 60%, preferably of atleast about 70%, more preferably of at least about 80%, more preferablyof at least about 90%, even more preferably of at least about 95%, evenmore preferably of at least about 99% to the nucleic acid sequenceaccording to SEQ ID NO. 26 (of the patent application WO2013/143700) ormore preferably to a corresponding RNA sequence, wherein, preferably,the fragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20% etc. of the full-length 5′-UTR.Preferably, the fragment exhibits a length of at least about 20nucleotides or more, preferably of at least about 30 nucleotides ormore, more preferably of at least about 40 nucleotides or more.Preferably, the fragment is a functional fragment as described herein.

In a further preferred embodiment, the at least one RNA of the inventivecomposition further comprises at least one 3′-UTR element whichcomprises or consists of a nucleic acid sequence derived from the 3′-UTRof a chordate gene, preferably a vertebrate gene, more preferably amammalian gene, most preferably a human gene, or from a variant of the3′-UTR of a chordate gene, preferably a vertebrate gene, more preferablya mammalian gene, most preferably a human gene.

The term ‘3’-UTR element′ refers to a nucleic acid sequence whichcomprises or consists of a nucleic acid sequence that is derived from a3′-UTR or from a variant of a 3′-UTR. A 3′-UTR element in the sense ofthe present invention may represent the 3′-UTR of an mRNA. Thus, in thesense of the present invention, preferably, a 3′-UTR element may be the3′-UTR of an mRNA, preferably of an artificial mRNA, or it may be thetranscription template for a 3′-UTR of an mRNA. Thus, a 3′-UTR elementpreferably is a nucleic acid sequence which corresponds to the 3′-UTR ofan mRNA, preferably to the 3′-UTR of an artificial mRNA, such as an mRNAobtained by transcription of a genetically engineered vector construct.Preferably, the 3′-UTR element fulfils the function of a 3′-UTR orencodes a sequence which fulfils the function of a 3′-UTR.

Preferably, the inventive mRNA comprises a 3′-UTR element which may bederivable from a gene that relates to an mRNA with an enhanced half-life(that provides a stable mRNA), for example a 3′-UTR element as definedand described below. Preferably, the 3′-UTR element, is a nucleic acidsequence derived from a 3′-UTR of a gene, which preferably encodes astable mRNA, or from a homolog, a fragment or a variant of said gene

In a particularly preferred embodiment, the 3′-UTR element comprises orconsists of a nucleic acid sequence which is derived from a 3′-UTR of agene selected from the group consisting of an albumin gene, an α-globingene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene,and a collagen alpha gene, such as a collagen alpha 1(I) gene, or from avariant of a 3′-UTR of a gene selected from the group consisting of analbumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylasegene, a lipoxygenase gene, and a collagen alpha gene, such as a collagenalpha 1(I) gene according to SEQ ID No. 1369-1390 of the patentapplication WO2013/143700 whose disclosure is incorporated herein byreference. In a particularly preferred embodiment, the 3′-UTR elementcomprises or consists of a nucleic acid sequence which is derived from a3′-UTR of an albumin gene, preferably a vertebrate albumin gene, morepreferably a mammalian albumin gene, most preferably a human albumingene, most preferably a human albumin gene according to SEQ ID NO. 10063(according SEQ ID No: 1369 of the patent application WO2013/143700). ThemRNA sequence may comprise or consist of a nucleic acid sequence whichis derived from the 3′-UTR of the human albumin gene according toGenBank Accession number NM_000477.5, or from a fragment or variantthereof.

In this context it is particularly preferred that the mRNA of theinventive composition comprises a 3′-UTR element comprising acorresponding RNA sequence derived from the nucleic acids according toSEQ ID No. 1369-1390 of the patent application WO2013/143700 or afragment, homolog or variant thereof.

Most preferably the 3′-UTR element comprises the nucleic acid sequencederived from a fragment of the human albumin gene (albumin7 3′UTR)according to SEQ ID NO. 10065 (according to SEQ ID No: 1376 of thepatent application WO2013/143700).

In this context it is particularly preferred that the 3′-UTR element ofthe at least one RNA of the inventive composition comprises or consistsof a corresponding RNA sequence of the nucleic acid sequence accordingto SEQ ID NO. 10066.

In another particularly preferred embodiment, the 3′-UTR elementcomprises or consists of a nucleic acid sequence which is derived from a3′-UTR of an α-globin gene, preferably a vertebrate α- or β-globin gene,more preferably a mammalian α- or β-globin gene, most preferably a humanα- or β-globin gene according to SEQ ID NO. 10055 (corresponding to SEQID No. 1370 of the patent application WO2013/143700 (3′-UTR of Homosapiens hemoglobin, alpha 1 (HBA1))), or according to SEQ ID NO. 10057(corresponding to SEQ ID No. 1371 of the patent applicationWO2013/143700 (3′-UTR of Homo sapiens hemoglobin, alpha 2 (HBA2))),and/or according to SEQ ID NO. 10059 (corresponding to SEQ ID No. 1372of the patent application WO2013/143700 (3′-UTR of Homo sapienshemoglobin, beta (HBB)).

For example, the 3′-UTR element may comprise or consist of the center,α-complex-binding portion of the 3′-UTR of an α-globin gene, accordingto SEQ ID NO. 10061 (corresponding to SEQ ID No. 1393 of the patentapplication WO2013/143700).

In this context it is particularly preferred that the 3′-UTR element ofthe RNA of the inventive composition comprises or consists of acorresponding RNA sequence of the nucleic acid sequence according to SEQID NO. 10062, according to the above or a homolog, a fragment or variantthereof.

The term ‘a nucleic acid sequence which is derived from the 3′-UTR of a[ . . . ] gene’ preferably refers to a nucleic acid sequence which isbased on the 3′-UTR sequence of a [ . . . ] gene or on a part thereof,such as on the 3′-UTR of an albumin gene, an α-globin gene, a β-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagenalpha gene, such as a collagen alpha 1(I) gene, preferably of an albumingene or on a part thereof. This term includes sequences corresponding tothe entire 3′-UTR sequence, i.e. the full length 3′-UTR sequence of agene, and sequences corresponding to a fragment of the 3′-UTR sequenceof a gene, such as an albumin gene, α-globin gene, β-globin gene,tyrosine hydroxylase gene, lipoxygenase gene, or collagen alpha gene,such as a collagen alpha 1(I) gene, preferably of an albumin gene.

The term ‘a nucleic acid sequence which is derived from a variant of the3′-UTR of a [ . . . ] gene’ preferably refers to a nucleic acid sequencewhich is based on a variant of the 3′-UTR sequence of a gene, such as ona variant of the 3′-UTR of an albumin gene, an α-globin gene, a β-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagenalpha gene, such as a collagen alpha 1(I) gene, or on a part thereof asdescribed above. This term includes sequences corresponding to theentire sequence of the variant of the 3′-UTR of a gene, i.e. the fulllength variant 3′-UTR sequence of a gene, and sequences corresponding toa fragment of the variant 3′-UTR sequence of a gene. A fragment in thiscontext preferably consists of a continuous stretch of nucleotidescorresponding to a continuous stretch of nucleotides in the full-lengthvariant 3′-UTR, which represents at least 20%, preferably at least 30%,more preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, and most preferably at least 90% of thefull-length variant 3′-UTR. Such a fragment of a variant, in the senseof the present invention, is preferably a functional fragment of avariant as described herein.

Preferably, the at least one 5′-UTR element and the at least one 3′-UTRelement act synergistically to increase protein production from the RNAof the inventive composition as described above.

Histone Stem Loop:

In a particularly preferred embodiment, the at least one RNA of theinventive composition comprises a histone stem-loop sequence/structure.Such histone stem-loop sequences are preferably selected from histonestem-loop sequences as disclosed in WO 2012/019780, whose disclosure isincorporated herewith by reference.

A histone stem-loop sequence, suitable to be used within the presentinvention, is preferably selected from at least one of the followingformulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

-   stem1 or stem2 bordering elements N₁₋₆ is a consecutive sequence of    1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more    preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each    N is independently from another selected from a nucleotide selected    from A, U, T, G and C, or a nucleotide analogue thereof;-   stem1 [N₀₋₂GN₃₋₅] is reverse complementary or partially reverse    complementary with element stem2, and is a consecutive sequence    between of 5 to 7 nucleotides;

wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1,more preferably of 1 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof;

wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of 4 to 5,more preferably of 4 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof, and

wherein G is guanosine or an analogue thereof, and may be optionallyreplaced by a cytidine or an analogue thereof, provided that itscomplementary nucleotide cytidine in stem2 is replaced by guanosine;

-   loop sequence [N₀₋₄(U/T)N₀₋₄] is located between elements stem1 and    stem2, and is a consecutive sequence of 3 to 5 nucleotides, more    preferably of 4 nucleotides;

wherein each N₀₋₄ is independent from another a consecutive sequence of0 to 4, preferably of 1 to 3, more preferably of 1 to 2 N, wherein eachN is independently from another selected from a nucleotide selected fromA, U, T, G and C or a nucleotide analogue thereof; and wherein U/Trepresents uridine, or optionally thymidine;

-   stem2 [N₃₋₅ CN₀₋₂] is reverse complementary or partially reverse    complementary with element stem1, and is a consecutive sequence    between of 5 to 7 nucleotides;

wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of 4 to 5,more preferably of 4 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof;

wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1,more preferably of 1 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G or C or a nucleotideanalogue thereof; and

wherein C is cytidine or an analogue thereof, and may be optionallyreplaced by a guanosine or an analogue thereof provided that itscomplementary nucleoside guanosine in stem1 is replaced by cytidine;

wherein stem1 and stem2 are capable of base pairing with each otherforming a reverse complementary sequence, wherein base pairing may occurbetween stem1 and stem2, e.g. by Watson-Crick base pairing ofnucleotides A and U/T or G and C or by non-Watson-Crick base pairinge.g. wobble base pairing, reverse Watson-Crick base pairing, Hoogsteenbase pairing, reverse Hoogsteen base pairing or are capable of basepairing with each other forming a partially reverse complementarysequence, wherein an incomplete base pairing may occur between stem1 andstem2, on the basis that one or more bases in one stem do not have acomplementary base in the reverse complementary sequence of the otherstem.

According to a further preferred embodiment of the first inventiveaspect, the inventive mRNA sequence may comprise at least one histonestem-loop sequence according to at least one of the following specificformulae (Ia) or (IIa):

formula (Ia) (stem-loop sequence without stem bordering elements):

formula (IIa) (stem-loop sequence with stem bordering elements):

wherein N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of thefirst aspect, the at least one RNA may comprise at least one histonestem-loop sequence according to at least one of the following specificformulae (Ib) or (IIb):

formula (Ib) (stem-loop sequence without stem bordering elements):

formula (IIb) (stem-loop sequence with stem bordering elements):

wherein N, C, G, T and U are as defined above.

A particular preferred histone stem-loop sequence is the sequenceaccording to SEQ ID No: 8.

More preferably the stem-loop sequence is the corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID NO: 9

Poly(A)

In a particularly preferred embodiment, the at least one RNA of theinventive composition comprises additionally to the coding regionencoding at least one peptide or protein as described above or afragment or variant thereof, a poly(A) sequence, also called poly-Atail, preferably at the 3′ terminus of the RNA. When present, such apoly(A) sequence comprises a sequence of about 25 to about 400 adenosinenucleotides, preferably a sequence of about 50 to about 400 adenosinenucleotides, more preferably a sequence of about 50 to about 300adenosine nucleotides, even more preferably a sequence of about 50 toabout 250 adenosine nucleotides, most preferably a sequence of about 60to about 250 adenosine nucleotides. In this context the term “about”refers to a deviation of ±10% of the value(s) it is attached to. Thispoly(A) sequence is preferably located 3′ of the coding region comprisedin the RNA according to the invention.

Preferably, the poly(A) sequence in at least one RNA of the compositionis derived from a DNA template by RNA in vitro transcription.Alternatively, the poly(A) sequence may also be obtained in vitro bycommon methods of chemical-synthesis without being necessarilytranscribed from a DNA-progenitor. Moreover, poly(A) sequences, orpoly(A) tails may be generated by enzymatic polyadenylation of the atleast one RNA using commercially available polyadenylation kits andcorresponding protocols known in the art.

Alternatively, the at least one RNA of the inventive compositionoptionally comprises a polyadenylation signal, which is defined hereinas a signal, which conveys polyadenylation to a (transcribed) RNA byspecific protein factors (e.g. cleavage and polyadenylation specificityfactor (CPSF), cleavage stimulation factor (CstF), cleavage factors Iand II (CF I and CF II), poly(A) polymerase (PAP)). In this context, aconsensus polyadenylation signal is preferred comprising the NN(U/T)ANAconsensus sequence. In a particularly preferred aspect, thepolyadenylation signal comprises one of the following sequences:AA(U/T)AAA or A(U/T)(U/T)AAA (wherein uridine is usually present in RNAand thymidine is usually present in DNA).

Poly(C)

According to a further preferred embodiment, the RNA of the inventivecomposition can be modified by a sequence of at least 10 cytosines,preferably at least 20 cytosines, more preferably at least 30 cytosines(so-called “poly(C) sequence”). Particularly, the RNA may contain apoly(C) sequence of typically about 10 to 200 cytosine nucleotides,preferably about 10 to 100 cytosine nucleotides, more preferably about10 to 70 cytosine nucleotides or even more preferably about 20 to 50 oreven 20 to 30 cytosine nucleotides. This poly(C) sequence is preferablylocated 3′ of the coding region, more preferably 3′ of an optionalpoly(A) sequence comprised in the RNA according to the presentinvention.

5′-Cap

According to another preferred embodiment of the invention, a modifiedRNA molecule as defined herein, can be modified by the addition of aso-called “5′ cap” structure, which preferably stabilizes the RNA asdescribed herein. A 5′-cap is an entity, typically a modified nucleotideentity, which generally “caps” the 5′-end of a mature mRNA. A 5′-cap maytypically be formed by a modified nucleotide, particularly by aderivative of a guanine nucleotide. Preferably, the 5′-cap is linked tothe 5′-terminus via a 5′-5′-triphosphate linkage. A 5′-cap may bemethylated, e.g. m7GpppN, wherein N is the terminal 5′ nucleotide of thenucleic acid carrying the 5′-cap, typically the 5′-end of an mRNA.m7GpppN is the 5′-cap structure, which naturally occurs in mRNAtranscribed by polymerase II and is therefore preferably not consideredas modification comprised in a modified RNA in this context.Accordingly, a modified RNA of the present invention may comprise am7GpppN as 5′-cap, but additionally the modified RNA typically comprisesat least one further modification as defined herein.

Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-cap structuresare regarded as at least one modification in this context.

Particularly preferred modified 5′-cap structures are cap1 (methylationof the ribose of the adjacent nucleotide of m7G), cap2 (additionalmethylation of the ribose of the 2nd nucleotide downstream of the m7G),cap3 (additional methylation of the ribose of the 3rd nucleotidedownstream of the m7G), cap4 (methylation of the ribose of the 4thnucleotide downstream of the m7G), ARCA (anti-reverse cap analogue,modified ARCA (e.g. phosphothioate modified ARCA), inosine,N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine.

Secretory Signal Sequence:

According to another particularly preferred embodiment, the at least oneRNA of the composition may additionally or alternatively encode asecretory signal peptide. Such secretory signal sequences are peptidestretches, which typically exhibit a length of about 15 to 30 aminoacids and are preferably located at the N-terminus of the encodedpeptide, without being limited thereto. Secretory signal sequences asdefined herein preferably allow the transport of the encoded peptide orprotein as encoded by the at least one coding sequence of the at leastone RNA of the composition into a defined cellular compartiment,preferably the cell surface, the endoplasmic reticulum (ER) or theendosomal-lysosomal compartiment. Examples of secretory signal sequencesas defined herein include, without being limited thereto, secretorysignal sequences of classical or non-classical MHC-molecules (e.g.signal sequences of MHC I and II molecules, e.g. of the MHC class Imolecule HLA-A*0201), secretory signal sequences of cytokines orimmunoglobulines as defined herein, secretory signal sequences of theinvariant chain of immunoglobulines or antibodies as defined herein,signal sequences of Lamp1, Tapasin, Erp57, Calretikulin, Calnexin, andfurther membrane associated proteins or of proteins associated with theendoplasmic reticulum (ER) or the endosomal-lysosomal compartiment.

Any of the above modifications regarding the coding sequence and/orregarding the RNA as defined above may be applied to the coding sequenceand/or the RNA of the composition of the present invention, and furtherto any RNA as used in the context of the present invention and may be,if suitable or necessary, be combined with each other in anycombination, provided, these combinations of modifications do notinterfere with each other in the respective at least one RNA. A personskilled in the art will be able to take his choice accordingly.

Production of mRNA and RNA

The RNA may be prepared using any method known in the art, includingsynthetic methods (chemical synthesis of RNA) such as e.g. solid phasesynthesis, as well as in vitro methods, such as RNA in vitrotranscription reactions.

Combinations:

According to the present invention it is particularly preferred tocombine RNA encoded peptides or proteins. In this context particularlypreferred are the following combinations:

-   -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one chemokine    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at at least one suicide gene product    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one immunogenic protein or        peptide    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one apoptosis inducer    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one angiogenesis inhibitor    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one heat shock protein    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one tumor antigen    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one β-catenin inhibitor    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one activator of the STING        pathway    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one checkpoint modulator    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one innate immune activator    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one antibody    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one decoy receptor    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one inhibitor of myeloid        derived suppressor cells (MDSCs)    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one IDO pathway inhibitor    -   RNA, preferably mRNA coding for at least one cytokine+RNA,        preferably mRNA coding for at least one protein or peptide that        bind inhibitors of apoptosis.

Furthermore, particularly preferred are the following embodiments:

-   -   RNA, preferably mRNA coding for IL-2 and/or RNA, preferably mRNA        coding for IL-12+mRNA coding for thymidine kinase (approach:        cytokines+suicide gene product)    -   RNA, preferably mRNA coding for IL-2 and/or RNA, preferably mRNA        coding for IL-12    -   RNA, preferably mRNA coding for IL-12 and/or RNA, preferably        mRNA coding for CD40L    -   RNA, preferably mRNA coding for IL-15 and/or RNA, preferably        mRNA coding for IL-12    -   RNA, preferably mRNA coding for IL-2+RNA, preferably mRNA coding        for Influenza NP protein    -   RNA, preferably mRNA coding for IL-2 and/or RNA, preferably mRNA        coding for IL-12+RNA, preferably mRNA coding for cytochrome        c/caspase 3 (cytokines+apoptosis induction)    -   RNA, preferably mRNA coding for CD40L+RNA, preferably mRNA        coding for IL-12+RNA, preferably mRNA coding for ΔRIGI

It has to be understood that the RNA molecules of the inventivecomposition may code for one or more different peptides or proteins(e.g. cytokines, chemokines, suicide gene products, immunogenic proteinsor peptides, apoptosis inducers, angiogenesis inhibitors, heat shockproteins, tumor antigens, β-catenin inhibitors, activators of the STINGpathway, checkpoint modulators, innate immune activators, antibodies,dominant negative receptors and decoy receptors, inhibitors of myeloidderived suppressor cells (MDSCs), IDO pathway inhibitors, and proteinsor peptides that bind inhibitors of apoptosis. as described above.Several RNA sequences may be combined in one inventive RNA containingcomposition. Moreover it is possible that the RNA sequence or sequencesof the inventive composition code for variants or fragments of the wildtype protein sequence or for one or more parts or fragments of the wildtype protein sequence or variants thereof.

Non Coding RNA

According to the invention the at least one RNA of the inventive RNAcontaining composition may comprise at least one non-coding RNA, whichis preferably selected from the group consisting of small interferingRNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes,aptamers, riboswitches, immunostimulating/immunostimulatory RNA RNA,transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA),small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA(piRNA).

Immunostimulatory/Immunostimulating RNA (isRNA):

Likewise, according to a further alternative, the at least one RNA ofthe inventive RNA containing composition is animmunostimulatory/immunostimulating RNA, which preferably elicits aninnate immune response. Such an immunostimulatory RNA may be any(double-stranded or single-stranded) RNA, e.g. a coding RNA, as definedherein. In a preferred embodiment, the immunostimulatory RNA is anon-coding RNA. Preferably, the immunostimulatory RNA may be asingle-stranded, a double-stranded or a partially double-stranded RNA,more preferably a single-stranded RNA, and/or a circular or linear RNA,more preferably a linear RNA. More preferably, the immunostimulatory RNAmay be a (linear) single-stranded RNA. Even more preferably, theimmunostimulatory RNA may be a (long) (linear) single-stranded)non-coding RNA. In this context it is particular preferred that theisRNA carries a triphosphate at its 5′-end which is the case for invitro transcribed RNA. An immunostimulatory RNA may also occur as ashort RNA oligonucleotide as defined herein.

An immunostimulatory RNA as used herein may furthermore be selected fromany class of RNA molecules, found in nature or being preparedsynthetically, and which can induce an innate immune response and maysupport an adaptive immune response induced by an antigen. In thiscontext, an immune response may occur in various ways. A substantialfactor for a suitable (adaptive) immune response is the stimulation ofdifferent T cell sub-populations. T-lymphocytes are typically dividedinto two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2(Th2) cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of the effectorproteins (cytokines) produced by them. Thus, Th1 cells assist thecellular immune response by activation of macrophages and cytotoxic Tcells. Th2 cells, on the other hand, promote the humoral immune responseby stimulation of B-cells for conversion into plasma cells and byformation of antibodies (e.g. against antigens). The Th1/Th2 ratio istherefore of great importance in the induction and maintenance of anadaptive immune response. In connection with the present invention, theTh1/Th2 ratio of the (adaptive) immune response is preferably shifted inthe direction towards the cellular response (Th1 response) and acellular immune response is thereby induced. According to one example,the innate immune system which may support an adaptive immune responsemay be activated by ligands of Toll-like receptors (TLRs). TLRs are afamily of highly conserved pattern recognition receptor (PRR)polypeptides that recognize pathogen-associated molecular patterns(PAMPs) and play a critical role in innate immunity in mammals.Currently at least thirteen family members, designated TLR1-TLR13(Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore,a number of specific TLR ligands have been identified. Furthermore, ithas been reported that ligands for certain TLRs include certain nucleicacid molecules and that certain types of RNA are immunostimulatory in asequence-independent or sequence-dependent manner, wherein these variousimmunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, orintracellular receptors such as RIG-I, MDA-5, etc.

Preferably, an immunostimulatory nucleic acid, preferably animmunostimulatory RNA (isRNA), as used herein, may comprise any RNAsequence known to be immunostimulatory, including, without being limitedthereto, RNA sequences representing and/or encoding ligands of TLRs,preferably selected from human family members TLR1-TLR10 or murinefamily members TLR1-TLR13, more preferably selected from (human) familymembers TLR1-TLR10, even more preferably from TLR7 and TLR8, ligands forintracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g.Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases:two parallel ways to trigger antiviral responses. Mol. Cell 22,561-569), or any other immunostimulatory RNA sequence. Furthermore,(classes of) immunostimulatory RNA molecules, used as a further compoundof the inventive vaccine, may include any other RNA capable of elicitingan immune response. Without being limited thereto, such animmunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA(tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such animmunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of5 to 50 or of 5 to 30 nucleotides.

An immunostimulatory RNA as used herein may furthermore be selected fromany class of RNA molecules, found in nature or being preparedsynthetically, and which can induce an innate immune response and maysupport an adaptive immune response induced by an antigen. In thiscontext, an immune response may occur in various ways. A substantialfactor for a suitable (adaptive) immune response is the stimulation ofdifferent T-cell sub-populations. T-lymphocytes are typically dividedinto two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2(Th2) cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of the effectorproteins (cytokines) produced by them. Thus, Th1 cells assist thecellular immune response by activation of macrophages and cytotoxicT-cells. Th2 cells, on the other hand, promote the humoral immuneresponse by stimulation of B-cells for conversion into plasma cells andby formation of antibodies (e.g. against antigens). The Th1/Th2 ratio istherefore of great importance in the induction and maintenance of anadaptive immune response. In connection with the present invention, theTh1/Th2 ratio of the (adaptive) immune response is preferably shifted inthe direction towards the cellular response (Th1 response) and acellular immune response is thereby induced. According to one example,the innate immune system which may support an adaptive immune response,may be activated by ligands of Toll-like receptors (TLRs). TLRs are afamily of highly conserved pattern recognition receptor (PRR)polypeptides that recognize pathogen-associated molecular patterns(PAMPs) and play a critical role in innate immunity in mammals.Currently at least thirteen family members, designated TLR1-TLR13(Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore,a number of specific TLR ligands have been identified. It was e.g. foundthat unmethylated bacterial DNA and synthetic analogs thereof (CpG DNA)are ligands for TLR9 (Hemmi H et al. (2000) Nature 408:740-5; Bauer S etal. (2001) Proc Natl Acad Sci USA 98, 9237-42). Furthermore, it has beenreported that ligands for certain TLRs include certain nucleic acidmolecules and that certain types of RNA are immunostimulatory in asequence-independent or sequence-dependent manner, wherein these variousimmunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, orintracellular receptors such as RIG-I, MDA-5, etc. E.g. Lipford et al.determined certain G,U-containing oligoribonucleotides asimmunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280). Theimmunostimulatory G,U-containing oligoribonucleotides described byLipford et al. were believed to be derivable from RNA sources includingribosomal RNA, transfer RNA, messenger RNA, and viral RNA.

According to a particularly preferred embodiment, such immunostimulatorynucleic acid sequences is preferably RNA preferably consisting of orcomprising a nucleic acid of the following formula (III) or (IV):

GIXmGn,  (formula (III))

wherein:

G is guanosine, uracil or an analogue of guanosine or uracil;

X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue ofthe above-mentioned nucleotides;

I is an integer from 1 to 40,

wherein

when I=1 G is guanosine or an analogue thereof,

when I>1 at least 50% of the nucleotides are guanosine or an analoguethereof;

m is an integer and is at least 3;

wherein

when m=3 X is uracil or an analogue thereof,

-   -   when m>3 at least 3 successive uracils or analogues of uracil        occur;

n is an integer from 1 to 40,

wherein

when n=1 G is guanosine or an analogue thereof,

when n>1 at least 50% of the nucleotides are guanosine or an analoguethereof.

CIXmCn,  (formula (IV))

wherein:

C is cytosine, uracil or an analogue of cytosine or uracil;

X is guanosine, uracil, adenosine, thymidine, cytosine or an analogue ofthe above-mentioned nucleotides;

I is an integer from 1 to 40,

wherein

when I=1 C is cytosine or an analogue thereof,

when I>1 at least 50% of the nucleotides are cytosine or an analoguethereof;

m is an integer and is at least 3;

wherein

when m=3 X is uracil or an analogue thereof,

when m>3 at least 3 successive uracils or analogues of uracil occur;

n is an integer from 1 to 40,

wherein

when n=1 C is cytosine or an analogue thereof,

when n>1 at least 50% of the nucleotides are cytosine or an analoguethereof.

The nucleic acids of formula (II) or (III), which may be used asimmunostimulatory RNA may be relatively short nucleic acid moleculeswith a typical length of approximately from 5 to 100 (but may also belonger than 100 nucleotides for specific embodiments, e.g. up to 200nucleotides), from 5 to 90 or from 5 to 80 nucleotides, preferably alength of approximately from 5 to 70, more preferably a length ofapproximately from 8 to 60 and, more preferably a length ofapproximately from 15 to 60 nucleotides, more preferably from 20 to 60,most preferably from 30 to 60 nucleotides. If the nucleic acid of theinventive nucleic acid cargo complex has a maximum length of e.g. 100nucleotides, m will typically be <=98. The number of nucleotides G inthe nucleic acid of formula (III) is determined by I or n. I and n,independently of one another, are each an integer from 1 to 40, whereinwhen I or n=1 G is guanosine or an analogue thereof, and when I or n>1at least 50% of the nucleotides are guanosine or an analogue thereof.For example, without implying any limitation, when I or n=4 GI or Gn canbe, for example, a GUGU, GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGGor GGGG, etc.; when I or n=5 GI or Gn can be, for example, a GGGUU,GGUGU, GUGGU, UGGGU, UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG, GGUGG,GUGGG, UGGGG, or GGGGG, etc.; etc. A nucleotide adjacent to Xm in thenucleic acid of formula (III) according to the invention is preferablynot a uracil. Similarly, the number of nucleotides C in the nucleic acidof formula (IV) according to the invention is determined by I or n. Iand n, independently of one another, are each an integer from 1 to 40,wherein when I or n=1 C is cytosine or an analogue thereof, and when Ior n>1 at least 50% of the nucleotides are cytosine or an analoguethereof. For example, without implying any limitation, when I or n=4, CIor Cn can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC,CUCC, UCCC or CCCC, etc.; when I or n=5 CI or Cn can be, for example, aCCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC, CUCUC, CCCCU, CCCUC,CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. A nucleotide adjacent to Xm inthe nucleic acid of formula (III) according to the invention ispreferably not a uracil. Preferably, for formula (II), when I or n>1, atleast 60%, 70%, 80%, 90% or even 100% of the nucleotides are guanosineor an analogue thereof, as defined above. The remaining nucleotides to100% (when guanosine constitutes less than 100% of the nucleotides) inthe flanking sequences G1 and/or Gn are uracil or an analogue thereof,as defined hereinbefore. Also preferably, I and n, independently of oneanother, are each an integer from 2 to 30, more preferably an integerfrom 2 to 20 and yet more preferably an integer from 2 to 15. The lowerlimit of I or n can be varied if necessary and is at least 1, preferablyat least 2, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10. Thisdefinition applies correspondingly to formula (III).

According to a particularly preferred embodiment, a nucleic acidaccording to any of formulas (III) or (IV) above, which may be used asimmunostimulatory RNA, may be selected from a sequence consisting orcomprising any of the following sequences SEQ ID NOs 298-381.

or from a sequence having at least 60%, 70%, 80%, 90%, or even 95%sequence identity with any of these sequences

According to a further particularly preferred embodiment, suchimmunostimulatory nucleic acid sequences particularly isRNA consist ofor comprise a nucleic acid of formula (V) or (VI):

(NuGIXmGnNv)a,  (formula (V))

wherein:

G is guanosine (guanine), uridine (uracil) or an analogue of guanosine(guanine) or uridine (uracil), preferably guanosine (guanine) or ananalogue thereof;

X is guanosine (guanine), uridine (uracil), adenosine (adenine),thymidine (thymine), cytidine (cytosine), or an analogue of thesenucleotides (nucleosides), preferably uridine (uracil) or an analoguethereof;

N is a nucleic acid sequence having a length of about 4 to 50,preferably of about 4 to 40, more preferably of about 4 to 30 or 4 to 20nucleic acids, each N independently being selected from guanosine(guanine), uridine (uracil), adenosine (adenine), thymidine (thymine),cytidine (cytosine) or an analogue of these nucleotides (nucleosides);

a is an integer from 1 to 20, preferably from 1 to 15, most preferablyfrom 1 to 10;

I is an integer from 1 to 40,

wherein when I=1, G is guanosine (guanine) or an analogue thereof,

-   -   when I>1, at least 50% of these nucleotides (nucleosides) are        guanosine (guanine) or an analogue thereof;

m is an integer and is at least 3;

wherein when m=3, X is uridine (uracil) or an analogue thereof, and

-   -   when m>3, at least 3 successive uridines (uracils) or analogues        of uridine (uracil) occur;

n is an integer from 1 to 40,

wherein when n=1, G is guanosine (guanine) or an analogue thereof,

-   -   when n>1, at least 50% of these nucleotides (nucleosides) are        guanosine (guanine) or an analogue thereof;

u,v may be independently from each other an integer from 0 to 50,

preferably wherein when u=0, v≧1, or

-   -   when v=0, u 1;

wherein the nucleic acid molecule of formula (IV) has a length of atleast 50 nucleotides, preferably of at least 100 nucleotides, morepreferably of at least 150 nucleotides, even more preferably of at least200 nucleotides and most preferably of at least 250 nucleotides.

(NuCIXmCnNv)a,  (formula (VI))

wherein:

C is cytidine (cytosine), uridine (uracil) or an analogue of cytidine(cytosine) or uridine (uracil), preferably cytidine (cytosine) or ananalogue thereof;

X is guanosine (guanine), uridine (uracil), adenosine (adenine),thymidine (thymine), cytidine (cytosine) or an analogue of theabove-mentioned nucleotides (nucleosides), preferably uridine (uracil)or an analogue thereof;

N is each a nucleic acid sequence having independent from each other alength of about 4 to 50, preferably of about 4 to 40, more preferably ofabout 4 to 30 or 4 to 20 nucleic acids, each N independently beingselected from guanosine (guanine), uridine (uracil), adenosine(adenine), thymidine (thymine), cytidine (cytosine) or an analogue ofthese nucleotides (nucleosides);

a is an integer from 1 to 20, preferably from 1 to 15, most preferablyfrom 1 to 10;

I is an integer from 1 to 40,

wherein when I=1, C is cytidine (cytosine) or an analogue thereof,

-   -   when I>1, at least 50% of these nucleotides (nucleosides) are        cytidine (cytosine) or an analogue thereof;

m is an integer and is at least 3;

wherein when m=3, X is uridine (uracil) or an analogue thereof,

-   -   when m>3, at least 3 successive uridines (uracils) or analogues        of uridine (uracil) occur;

n is an integer from 1 to 40,

wherein when n=1, C is cytidine (cytosine) or an analogue thereof,

-   -   when n>1, at least 50% of these nucleotides (nucleosides) are        cytidine (cytosine) or an analogue thereof.

u, v may be independently from each other an integer from 0 to 50,

preferably wherein when u=0, v≧1, or

-   -   when v=0, u≧1;

wherein the nucleic acid molecule of formula (V) according to theinvention has a length of at least 50 nucleotides, preferably of atleast 100 nucleotides, more preferably of at least 150 nucleotides, evenmore preferably of at least 200 nucleotides and most preferably of atleast 250 nucleotides.

For formula (VI), any of the definitions given above for elements N(i.e. Nu and Nv) and X (Xm), particularly the core structure as definedabove, as well as for integers a, I, m, n, u and v, similarly apply toelements of formula (VI) correspondingly, wherein in formula (VI) thecore structure is defined by CIXmCn. The definition of borderingelements Nu and Nv is identical to the definitions given above for Nuand Nv.

According to a very particularly preferred embodiment, the nucleic acidmolecule, preferably immunostimulating RNA according to formula (V) maybe selected from e.g. any of the sequences according to SEQ ID NOs382-395 or from a sequence having at least 60%, 70%, 80%, 90%, or even95% sequence identity with any of these sequences.

In this context particularly preferred are immunostimulating RNAsaccording to SEQ ID NOs 5, 394 and 10072.

R2025:

(SEQ ID NO: 5 and 394)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAG 

R3630:

(SEQ ID NO. 10072) GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUGCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAG.

According to another very particularly preferred embodiment, the nucleicacid molecule according to formula (VI) may be selected from e.g. any ofthe sequences according to SEQ ID NO 396 or 397, or from a sequencehaving at least 60%, 70%, 80%, 90%, or even 95% sequence identity withany of these sequences.

All modifications disclosed in the context of coding RNA may also beapplied in the context of non-coding RNA if applicable.

Combination of Coding and Non-Coding RNA

In particularly preferred embodiments the inventive RNA containingcomposition comprises at least one RNA encoding at least one peptide orprotein and at least one non-coding RNA as defined above, preferably atleast one immunostimulating RNA.

Particularly preferred are the following embodiments:

-   -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one cytokine,        preferably IL-2, IL-12, IL-15 or CD40L    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one chemokine    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one suicide        gene product, preferably Herpes simplex virus thymidine kinase    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one        immunogenic protein or peptide, preferably Influenza NP protein    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one apoptosis        inducer, preferably cytochrome c or caspase 3    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one        angiogenesis inducer    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one heat shock        protein    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one tumor        antigen    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one β-catenin        inhibitor    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one activator        of the STING pathway    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one checkpoint        modulator, preferably an antibody directed against PD-1, PD-L1        or CTLA4    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one innate        immune activator, preferably a constitutive active variant of        RIG-1    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one antibody    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one decoy        receptor, preferably a soluble PD-1 receptor    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one inhibitor        of myeloid derived suppressor cells (MDSCs)    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one IDO        pathway inhibitor    -   Immostimulating RNA preferably according to SEQ ID Nos XY or        YY+RNA, preferably mRNA coding for at least one protein or        peptide that bind inhibitors of apoptosis.

More particularly preferred are the following embodiments:

-   -   Immostimulating RNA preferably according SEQ ID Nos 5, 394, or        10072+RNA, preferably mRNA coding for at least one cytokine,        preferably IL-2, IL-12, IL-15 or CD40L+RNA, preferably mRNA        coding for at least one immunogenic protein or peptide,        preferably Influenza NP protein    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one cytokine,        preferably IL-2, IL-12, IL-15 or CD40L+RNA, preferably mRNA        coding for at least one innate immune activator, preferably a        constitutive active variant of RIG-1    -   Immostimulating RNA preferably according to SEQ ID Nos 5, 394,        or 10072+RNA, preferably mRNA coding for at least one cytokine,        preferably IL-2, IL-12, or IL-15, +RNA, preferably mRNA coding        for at least one further cytokine, preferably CD40L.

Formulation and Complexation

The at least one RNA of the inventive composition may be administerednaked without being associated with any further vehicle, carrier,transfection or complexation agent.

In a preferred embodiment, the RNA of the inventive composition isformulated together with further compounds for increasing thetransfection efficiency and/or the immunostimulatory properties of theRNA. Such compounds are termed herein carriers, vehicles, transfectionor complexation agents. Preferably, the RNA is formulated together withone or more cationic or polycationic compounds, preferably with cationicor polycationic polymers, cationic or polycationic peptides or proteins,cationic or polycationic polysaccharides, cationic or polycationiclipids and/or with a polymeric carrier. Such cationic or polycationicpolymers, cationic or polycationic peptides or proteins, cationic orpolycationic polysaccharides, cationic or polycationic lipids orpolymeric carriers are useful as carriers, vehicles, transfection orcomplexation agents of nucleic acids in the context of the presentinvention. Accordingly, in a further embodiment of the invention it ispreferred that the at least one RNA or any other nucleic acid comprisedin the inventive composition is associated with or complexed with acationic or polycationic compound or a polymeric carrier, optionally ina weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1(w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), evenmore preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)to about 2:1 (w/w) of RNA or nucleic acid to cationic or polycationiccompound and/or with a polymeric carrier; or optionally in anitrogen/phosphate ratio of RNA or nucleic acid to cationic orpolycationic compound and/or polymeric carrier in the range of about0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and mostpreferably in a range of about 0.5-1 or 0.7-1, and even most preferablyin a range of about 0.3-0.9 or 0.5-0.9.

The ratio of the at least one RNA as described above, and the cationicor polycationic compound, may be calculated on the basis of thenitrogen/phosphate ratio (N/P-ratio) of all these components. In thecontext of the present invention, an N/P-ratio is preferably in therange of about 0.01-4, 0.01-2, 0.1-2 or 0.1-1.5 regarding the ratio ofnucleic acids:cationic or polycationic peptide contained in theinventive vaccine, and most preferably in the range of about 0.1-1. Suchan N/P ratio is preferably designed to provide good transfectionproperties in vivo and transport into and through cell membranes.Preferably, for this purpose, cationic or polycationic compound and/orpolymeric carriers as used herein, are based on peptide sequences.

Cationic or polycationic compounds, being particularly preferred agentsin this context include protamine, nucleoline, spermine or spermidine,or other cationic peptides or proteins, such as poly-L-lysine (PLL),poly-arginine, basic polypeptides, cell penetrating peptides (CPPs),including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides,Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex),MAP, KALA or protein transduction domains (PTDs), PpT620, proline-richpeptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s),Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIsI, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, or histones.

In this context protamine is particularly preferred.

Additionally, preferred cationic or polycationic proteins or peptidesmay be selected from the following proteins or peptides having thefollowing total formula (VII):

(Arg)_(I);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x),  (formula (VII)

wherein I+m+n+o+x=8-15, and I, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred cationic peptides in this context are e.g. Arg₇,Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. In thiscontext the disclosure of WO 2009/030481 is incorporated herewith byreference.

A polymeric carrier used according to the invention might be a polymericcarrier formed by disulfide-crosslinked cationic components.

According to a further particularly preferred embodiment, cationic orpolycationic peptides or proteins of the polymeric carrier, having theempirical sum formula (VII) as shown above and which comprise or areadditionally modified to comprise at least one —SH moeity, may be,without being restricted thereto, selected from the subgroup consistingof generic formulas Arg7 (also termed as R7), Arg9 (also termed R9),Arg12 (also termed as R12).

According to a one further particularly preferred embodiment, thecationic or polycationic peptide or protein of the polymeric carrier,when defined according to formula {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x}(formula (VII)) as shown above and which comprise or are additionallymodified to comprise at least one —SH moeity, may be, without beingrestricted thereto, selected from subformula (VIIa):

{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa′)x(Cys)y}  formula (VIIa)

wherein (Arg)I;(Lys)m;(His)n;(Orn)o; and x are as defined herein, Xaa′is any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His, Orn or Cys and y is anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80 and81-90, provided that the overall content of Arg (Arginine), Lys(Lysine), His (Histidine) and Orn (Ornithine) represents at least 10% ofall amino acids of the oligopeptide.

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x (formula (VII)) as shown above,comprises or has been modified with at least one cysteine as —SH moietyin the above meaning such that the cationic or polycationic peptide ascationic component carries at least one cysteine, which is capable toform a disulfide bond with other components of the polymeric carrier.

Exemplary examples may comprise any of the following sequences:

Cys(Arg7), Cys(Arg8), Cys(Arg9), Cys(Arg10), Cys(Arg11), Cys(Arg12),Cys(Arg13), Cys(Arg14), Cys(Arg15), Cys(Arg16), Cys(Arg17), Cys(Arg18),Cys(Arg19), Cys(Arg20).

According to another particularly preferred embodiment, the cationic orpolycationic peptide or protein of the polymeric carrier, when definedaccording to formula {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula(VII)) as shown above, may be, without being restricted thereto,selected from subformula (VIIb):

Cys1{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys2  (formula (VIIb))

wherein empirical formula {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula(VII)) is as defined herein and forms a core of an amino acid sequenceaccording to (semiempirical) formula (I) and wherein Cys1 and Cys2 areCysteines proximal to, or terminal to(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x. Exemplary examples may comprise anyof the above sequences flanked by two Cys and following sequences:

Cys(Arg7)Cys, Cys(Arg8)Cys, Cys(Arg9)Cys, Cys(Arg10)Cys, Cys(Arg11)Cys,Cys(Arg12)Cys, Cys(Arg13)Cys, Cys(Arg14)Cys, Cys(Arg15)Cys,Cys(Arg16)Cys, Cys(Arg17)Cys, Cys(Arg18)Cys, Cys(Arg19)Cys,Cys(Arg20)Cys (SEQ ID NOs: 10-23):

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x (formula (VII)) as shown above, hasbeen modified with at least two cysteines as —SH moieties in the abovemeaning such that the cationic or polycationic peptide of the inventivepolymeric carrier cargo complex as cationic component carries at leasttwo (terminal) cysteines, which are capable to form a disulfide bondwith other components of the polymeric carrier.

In a preferred embodiment, the polymeric carrier is formed by, comprisesor consists of the peptide CysArg12Cys (SEQ ID NO: 15) or CysArg12(CRRRRRRRRRRRR).

According to a second alternative, at least one cationic (orpolycationic) component of the polymeric carrier may be selected frome.g. any (non-peptidic) cationic or polycationic polymer suitable inthis context, provided that this (non-peptidic) cationic or polycationicpolymer exhibits or is modified to exhibit at least one —SH-moiety,which provide for a disulfide bond linking the cationic or polycationicpolymer with another component of the polymeric carrier as definedherein. Thus, likewise as defined herein, the polymeric carrier maycomprise the same or different cationic or polycationic polymers.

In the specific case that the cationic component of the polymericcarrier comprises a (non-peptidic) cationic or polycationic polymer thecationic properties of the (non-peptidic) cationic or polycationicpolymer may be determined upon its content of cationic charges whencompared to the overall charges of the components of the cationicpolymer. Preferably, the content of cationic charges in the cationicpolymer at a (physiological) pH as defined herein is at least 10%, 20%,or 30%, preferably at least 40%, more preferably at least 50%, 60% or70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%,99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to90%, more preferably in the range of about 30% to 100%, even preferablyin the range of about 50% to 100%, e.g. 50, 60, 70, 80%, 90% or 100%, orin a range formed by any two of the afore mentioned values, provided,that the content of all charges, e.g. positive and negative charges at a(physiological) pH as defined herein, in the entire cationic polymer is100%.

Preferably, the (non-peptidic) cationic component of the polymericcarrier represents a cationic or polycationic polymer, typicallyexhibiting a molecular weight of about 0.1 or 0.5 kDa to about 100 kDa,preferably of about 1 kDa to about 75 kDa, more preferably of about 5kDa to about 50 kDa, even more preferably of about 5 kDa to about 30kDa, or a molecular weight of about 10 kDa to about 50 kDa, even morepreferably of about 10 kDa to about 30 kDa. Additionally, the(non-peptidic) cationic or polycationic polymer typically exhibits atleast one —SH-moiety, which is capable to form a disulfide linkage uponcondensation with either other cationic components or other componentsof the polymeric carrier as defined herein.

In the above context, the (non-peptidic) cationic component of thepolymeric carrier may be selected from acrylates, modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), chitosanes,aziridines or 2-ethyl-2-oxazoline (forming oligo ethylenimines ormodified oligoethylenimines), polymers obtained by reaction ofbisacrylates with amines forming oligo beta aminoesters or poly amidoamines, or other polymers like polyesters, polycarbonates, etc. Eachmolecule of these (non-peptidic) cationic or polycationic polymerstypically exhibits at least one —SH-moiety, wherein these at least one—SH-moiety may be introduced into the (non-peptidic) cationic orpolycationic polymer by chemical modifications, e.g. using imonothiolan,3-thio propionic acid or introduction of —SH-moieties containing aminoacids, such as cysteine or any further (modified) amino acid. Such—SH-moieties are preferably as already defined above.

The disulfide-crosslinked cationic components may be the same ordifferent from each other. The polymeric carrier can also containfurther components. It is also particularly preferred that the polymericcarrier used according to the present invention comprises mixtures ofcationic peptides, proteins or polymers and optionally furthercomponents as defined herein, which are crosslinked by disulfide bondsas described herein. In this context the disclosure of WO 2012/013326 isincorporated herewith by reference.

In this context the cationic components, which form basis for thepolymeric carrier by disulfide-crosslinkage, are typically selected fromany suitable cationic or polycationic peptide, protein or polymersuitable for this purpose, particular any cationic or polycationicpeptide, protein or polymer capable to complex an RNA or a nucleic acidas defined according to the present invention, and thereby preferablycondensing the RNA or the nucleic acid. The cationic or polycationicpeptide, protein or polymer, is preferably a linear molecule, however,branched cationic or polycationic peptides, proteins or polymers mayalso be used.

Every disulfide-crosslinking cationic or polycationic protein, peptideor polymer of the polymeric carrier, which may be used to complex theRNA of the inventive composition or any further nucleic acid comprisedin the inventive composition contains at least one —SH moiety, mostpreferably at least one cysteine residue or any further chemical groupexhibiting an —SH moiety, capable to form a disulfide linkage uponcondensation with at least one further cationic or polycationic protein,peptide or polymer as cationic component of the polymeric carrier asmentioned herein.

As defined above, the polymeric carrier, which may be used to complexthe RNA of the inventive composition or any further nucleic acidcomprised in the inventive composition may be formed bydisulfide-crosslinked cationic (or polycationic) components.

Nucleic acids complexed with such polymeric carriers are also termedherein as “polymeric carrier cargo complexes”.

In this context it is particularly preferred that if immunostimulatingRNA is used in the context of the present invention that thisimmunostimulating RNA is complexed with a polymeric carrier as definedabove. Preferably, the immunostimulating RNA, (e.g. comprising an RNAsequence according to any of formulae III-VI), most preferablycomprising an RNA sequence according to SEQ ID NOs. 5, 394, or 10072, iscomplexed with a polymeric carrier comprising or formed bydisulfide-crosslinked peptides according to formula VII, VIIa or VIIb,preferably a polymeric carrier formed by Cys(Arg12)Cys or Cys(Arg12).Such a particularly preferred embodiment is termed herein also as“RNAdjuvant”.

In a further particular embodiment, the polymeric carrier which may beused to complex the RNA or any further nucleic acid comprised in theinventive composition may be selected from a polymeric carrier moleculeaccording to generic formula (VIII):

L-P¹—S—[S—P²—S]_(n)—S—P³-L  formula (VIII)

wherein,

-   P¹ and P³ are different or identical to each other and represent a    linear or branched hydrophilic polymer chain, each P¹ and P³    exhibiting at least one —SH-moiety, capable to form a disulfide    linkage upon condensation with component P², or alternatively with    (AA), (AA)_(x), or [(AA)_(x)]_(z) if such components are used as a    linker between P¹ and P² or P³ and P² and/or with further components    (e.g. (AA), (AA)_(x), [(AA)_(x)]_(z), or L), the linear or branched    hydrophilic polymer chain selected independent from each other from    polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide,    poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl    L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),    hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the    hydrophilic polymer chain exhibits a molecular weight of about 1 kDa    to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or more    preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to about    25 kDa or 5 kDa to about 10 kDa;-   P² is a cationic or polycationic peptide or protein, e.g. as defined    above for the polymeric carrier formed by disulfide-crosslinked    cationic components, and preferably having a length of about 3 to    about 100 amino acids, more preferably having a length of about 3 to    about 50 amino acids, even more preferably having a length of about    3 to about 25 amino acids, e.g. a length of about 3 to 10, 5 to 15,    10 to 20 or 15 to 25 amino acids, more preferably a length of about    5 to about 20 and even more preferably a length of about 10 to about    20; or    -   is a cationic or polycationic polymer, e.g. as defined above for        the polymeric carrier formed by disulfide-crosslinked cationic        components, typically having a molecular weight of about 0.5 kDa        to about 30 kDa, including a molecular weight of about 1 kDa to        about 20 kDa, even more preferably of about 1.5 kDa to about 10        kDa, or having a molecular weight of about 0.5 kDa to about 100        kDa, including a molecular weight of about 10 kDa to about 50        kDa, even more preferably of about 10 kDa to about 30 kDa;    -   each P² exhibiting at least two —SH-moieties, capable to form a        disulfide linkage upon condensation with further components P²        or component(s) P¹ and/or P³ or alternatively with further        components (e.g. (AA), (AA)_(x), or [(AA)_(x)]_(z));-   —S—S— is a (reversible) disulfide bond (the brackets are omitted for    better readability), wherein S preferably represents sulphur or a    —SH carrying moiety, which has formed a (reversible) disulfide bond.    The (reversible) disulfide bond is preferably formed by condensation    of —SH-moieties of either components P¹ and P², P² and P², or P² and    P³, or optionally of further components as defined herein (e.g. L,    (AA), (AA)_(x), [(AA)_(x)]_(z), etc); The —SH-moiety may be part of    the structure of these components or added by a modification as    defined below;-   L is an optional ligand, which may be present or not, and may be    selected independent from the other from RGD, Transferrin, Folate, a    signal peptide or signal sequence, a localization signal or    sequence, a nuclear localization signal or sequence (NLS), an    antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand    of a receptor (e.g. cytokines, hormones, growth factors etc), small    molecules (e.g. carbohydrates like mannose or galactose or synthetic    ligands), small molecule agonists, inhibitors or antagonists of    receptors (e.g. RGD peptidomimetic analogues), or any further    protein as defined herein, etc.;-   n is an integer, typically selected from a range of about 1 to 50,    preferably from a range of about 1, 2 or 3 to 30, more preferably    from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1,    2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or    a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of    about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a    range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of    about 6 to 11 or 7 to 10. Most preferably, n is in a range of about    1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3,    or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of    about 1, 2, or 3 to 7.

In this context the disclosure of WO 2011/026641 and WO 2012/116811 isincorporated herewith by reference. Each of hydrophilic polymers P¹ andP³ typically exhibits at least one —SH-moiety, wherein the at least one—SH-moiety is capable to form a disulfide linkage upon reaction withcomponent P² or with component (AA) or (AA)_(x), if used as linkerbetween P¹ and P² or P³ and P² as defined below and optionally with afurther component, e.g. L and/or (AA) or (AA)_(x), e.g. if two or more—SH-moieties are contained. The following subformulae “P¹—S—S—P²” and“P²—S—S—P³” within the generic formula above, wherein any of S, P¹ andP³ are as defined herein, typically represent a situation, wherein one—SH-moiety of hydrophilic polymers P¹ and P³ was condensed with one—SH-moiety of component P² of the generic formula above, wherein bothsulphurs of these —SH-moieties form a disulfide bond —S—S—. These—SH-moieties are typically provided by each of the hydrophilic polymersP¹ and P³, e.g. via an internal cysteine or any further (modified) aminoacid or compound which carries a —SH moiety. Accordingly, thesubformulae “P¹—S—S—P²” and “P²—S—S—P³” may also be written as“P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”, if the —SH-moiety is provided by acysteine, wherein the term Cys-Cys represents two cysteines coupled viaa disulfide bond, not via a peptide bond. In this case, the term “—S—S—”in these formulae may also be written as “—S-Cys”, as “-Cys-S” or as“-Cys-Cys-”. In this context, the term “-Cys-Cys-” does not represent apeptide bond but a linkage of two cysteines via their —SH-moieties toform a disulfide bond. Accordingly, the term “-Cys-Cys-” also may beunderstood generally as “-(Cys-S)—(S-Cys)-”, wherein in this specificcase S indicates the sulphur of the —SH-moiety of cysteine. Likewise,the terms “—S-Cys” and “-Cys-S” indicate a disulfide bond between a —SHcontaining moiety and a cysteine, which may also be written as“—S—(S-Cys)” and “-(Cys-S)—S”. Alternatively, the hydrophilic polymersP¹ and P³ may be modified with a —SH moiety, preferably via a chemicalreaction with a compound carrying a —SH moiety, such that each of thehydrophilic polymers P¹ and P³ carries at least one such —SH moiety.Such a compound carrying a —SH moiety may be e.g. an (additional)cysteine or any further (modified) amino acid, which carries a —SHmoiety. Such a compound may also be any non-amino compound or moiety,which contains or allows to introduce a —SH moiety into hydrophilicpolymers P¹ and P³ as defined herein. Such non-amino compounds may beattached to the hydrophilic polymers P¹ and P³ of the polymeric carriervia chemical reactions or binding of compounds, e.g. by binding of a3-thio propionic acid or thioimolane, by amide formation (e.g.carboxylic acids, sulphonic acids, amines, etc), by Michael addition(e.g maleinimide moieties, unsatured carbonyls, etc), by click chemistry(e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes oralkines), imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components. Aparticularly preferred PEG derivate in this context isalpha-Methoxy-omega-mercapto poly(ethylene glycol). In each case, theSH-moiety, e.g. of a cysteine or of any further (modified) amino acid orcompound, may be present at the terminal ends or internally at anyposition of hydrophilic polymers P¹ and P³. As defined herein, each ofhydrophilic polymers P¹ and P³ typically exhibits at least one—SH-moiety preferably at one terminal end, but may also contain two oreven more —SH-moieties, which may be used to additionally attach furthercomponents as defined herein, preferably further functional peptides orproteins e.g. a ligand, an amino acid component (AA) or (AA)_(x),antibodies, cell penetrating peptides or enhancer peptides (e.g. TAT,KALA), etc.

As defined above, ligands (L), may be optionally used in the polymericcarrier molecule according to generic formula (VIII), e.g. for directionof the inventive carrier polymer and its entire “cargo” (the adjuvantcomponent and/or the antigen of the inventive composition or vaccinecomposition) into specific cells. They may be selected independent fromthe other from RGD, Transferrin, Folate, a signal peptide or signalsequence, a localization signal or sequence, a nuclear localizationsignal or sequence (NLS), an antibody, a cell penetrating peptide (CPP),(e.g. TAT, KALA), a ligand of a receptor (e.g. cytokines, hormones,growth factors etc), small molecules (e.g. carbohydrates like mannose orgalactose or synthetic ligands), small molecule agonists, inhibitors orantagonists of receptors (e.g. RGD peptidomimetic analogues) or any suchmolecule as further defined below, etc. Particularly preferred are cellpenetrating peptides (CPPs), which induce a pH-mediated conformationalchange in the endosome and lead to an improved release of the inventivepolymeric carrier (in complex with a nucleic acid) from the endosome byinsertion into the lipid layer of the liposome. Such called CPPs orcationic peptides for transportation, may include, without being limitedthereto protamine, nucleoline, spermine or spermidine, poly-L-lysine(PLL), basic polypeptides, poly-arginine, chimeric CPPs, such asTransportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat(HIV), Tat-derived peptides, oligoarginines, members of the penetratinfamily, e.g. Penetratin, Antennapedia-derived peptides (particularlyfrom Drosophila antennapedia), pAntp, pIsI, etc., antimicrobial-derivedCPPs e.g. Buforin-2, Bac715-24, Syn B, SynB(1), pVEC, hCT-derivedpeptides, SAP, MAP, PpTG20, Proline-rich peptides, Loligomers,Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin,poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides,Pestivirus Erns, HSV, VP22 (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs, PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitoninpeptide(s), etc. Particularly preferred in this context is mannose asligand to target antigen presenting cells which carries on their cellmembrane mannose receptors. In a further preferred aspect of the firstembodiment of the present invention galactose as optional ligand can beused to target hepatocytes. Such ligands may be attached to component P¹and/or P³ by reversible disulfide bonds as defined below or by any otherpossible chemical attachment, e.g. by amide formation (e.g. carboxylicacids, sulphonic acids, amines, etc), by Michael addition (e.g.maleinimide moieties, α,β unsatured carbonyls, etc), by click chemistry(e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes oralkines), imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components.

In the context of formula (VIII) of the present invention components P¹and P³ represent a linear or branched hydrophilic polymer chain,containing at least one —SH-moiety, each P¹ and P³ independentlyselected from each other, e.g. from polyethylene glycol (PEG),poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethylphosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkylL-glutamine). P¹ and P³ may be identical or different to each other.Preferably, each of hydrophilic polymers P¹ and P³ exhibits a molecularweight of about 1 kDa to about 100 kDa, preferably of about 1 kDa toabout 75 kDa, more preferably of about 5 kDa to about 50 kDa, even morepreferably of about 5 kDa to about 25 kDa. Additionally, each ofhydrophilic polymers P¹ and P³ typically exhibits at least one—SH-moiety, wherein the at least one —SH-moiety is capable to form adisulfide linkage upon reaction with component P² or with component (AA)or (AA)_(x), if used as linker between P¹ and P² or P³ and P² as definedbelow and optionally with a further component, e.g. L and/or (AA) or(AA)_(x), e.g. if two or more —SH-moieties are contained. The followingsubformulae “P¹—S—S—P²” and “P²—S—S—P³” within generic formula (VII)above (the brackets are omitted for better readability), wherein any ofS, P¹ and P³ are as defined herein, typically represent a situation,wherein one —SH-moiety of hydrophilic polymers P¹ and P³ was condensedwith one —SH-moiety of component P² of generic formula (VII) above,wherein both sulphurs of these —SH-moieties form a disulfide bond —S—S—as defined herein in formula (VII). These —SH-moieties are typicallyprovided by each of the hydrophilic polymers P¹ and P³, e.g. via aninternal cysteine or any further (modified) amino acid or compound whichcarries a —SH moiety. Accordingly, the subformulae “P¹—S—S—P²” and“P²—S—S—P³” may also be written as “P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”,if the —SH-moiety is provided by a cysteine, wherein the term Cys-Cysrepresents two cysteines coupled via a disulfide bond, not via a peptidebond. In this case, the term “—S—S—” in these formulae may also bewritten as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, theterm “-Cys-Cys-” does not represent a peptide bond but a linkage of twocysteines via their —SH-moieties to form a disulfide bond. Accordingly,the term “-Cys-Cys-” also may be understood generally as“-(Cys-S)—(S-Cys)-”, wherein in this specific case S indicates thesulphur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and“-Cys-S” indicate a disulfide bond between a —SH containing moiety and acysteine, which may also be written as “—S—(S-Cys)” and “-(Cys-S)—S”.Alternatively, the hydrophilic polymers P¹ and P³ may be modified with a—SH moiety, preferably via a chemical reaction with a compound carryinga —SH moiety, such that each of the hydrophilic polymers P¹ and P³carries at least one such —SH moiety. Such a compound carrying a —SHmoiety may be e.g. an (additional) cysteine or any further (modified)amino acid, which carries a —SH moiety. Such a compound may also be anynon-amino compound or moiety, which contains or allows to introduce a—SH moiety into hydrophilic polymers P¹ and P³ as defined herein. Suchnon-amino compounds may be attached to the hydrophilic polymers P¹ andP³ of formula (VII) of the polymeric carrier according to the presentinvention via chemical reactions or binding of compounds, e.g. bybinding of a 3-thio propionic acid or thioimolane, by amide formation(e.g. carboxylic acids, sulphonic acids, amines, etc), by Michaeladdition (e.g maleinimide moieties, α,β unsatured carbonyls, etc), byclick chemistry (e.g. azides or alkines), by alkene/alkine methatesis(e.g. alkenes or alkines), imine or hydrozone formation (aldehydes orketons, hydrazins, hydroxylamins, amines), complexation reactions(avidin, biotin, protein G) or components which allow S_(n)-typesubstitution reactions (e.g halogenalkans, thiols, alcohols, amines,hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) orother chemical moieties which can be utilized in the attachment offurther components. A particularly preferred PEG derivate in thiscontext is alpha-Methoxy-omega-mercapto poly(ethylene glycol). In eachcase, the SH-moiety, e.g. of a cysteine or of any further (modified)amino acid or compound, may be present at the terminal ends orinternally at any position of hydrophilic polymers P¹ and P³. As definedherein, each of hydrophilic polymers P¹ and P³ typically exhibits atleast one —SH-moiety preferably at one terminal end, but may alsocontain two or even more —SH-moieties, which may be used to additionallyattach further components as defined herein, preferably furtherfunctional peptides or proteins e.g. a ligand, an amino acid component(AA) or (AA)_(x), antibodies, cell penetrating peptides or enhancerpeptides (e.g. TAT, KALA), etc.

According to one preferred alternative, such further functional peptidesor proteins may comprise so called cell penetrating peptides (CPPs) orcationic peptides for transportation. Particularly preferred are CPPs,which induce a pH-mediated conformational change in the endosome andlead to an improved release of the inventive polymeric carrier (incomplex with a nucleic acid) from the endosome by insertion into thelipid layer of the liposome. Such called cell penetrating peptides(CPPs) or cationic peptides for transportation, may include, withoutbeing limited thereto protamine, nucleoline, spermine or spermidine,poly-L-lysine (PLL), basic polypeptides, poly-arginine, chimeric CPPs,such as Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1Tat (HIV), Tat-derived peptides, oligoarginines, members of thepenetratin family, e.g. Penetratin, Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIsI, etc.,antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, Syn B, SynB(1),pVEC, hCT-derived peptides, SAP, MAP, PpTG20, Proline-rich peptides,Loligomers, Arginine-rich peptides, Calcitonin-peptides, FGF,Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived oranalog peptides, Pestivirus Erns, HSV, VP22 (Herpes simplex), MAP, KALAor protein transduction domains (PTDs, PpT620, prolin-rich peptides,arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers,Calcitonin peptide(s), etc.

According to a further preferred embodiment of the present invention,each of hydrophilic polymers P¹ and P³ of formula (VIII) of thepolymeric carrier used according to the present invention may alsocontain at least one further functional moiety, which allows attachingfurther components as defined herein, e.g. a ligand as defined above, orfunctionalities which allow the attachment of further components, e.g.by amide formation (e.g. carboxylic acids, sulphonic acids, amines,etc), by Michael addition (e.g maleinimide moieties, unsaturedcarbonyls, etc), by click chemistry (e.g. azides or alkines), byalkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozoneformation (aldehydes or ketons, hydrazins, hydroxylamins, amines),complexation reactions (avidin, biotin, protein G) or components whichallow S_(n)-type substitution reactions (e.g halogenalkans, thiols,alcohols, amines, hydrazines, hydrazides, sulphonic acid esters,oxyphosphonium salts) or other chemical moieties which can be utilizedin the attachment of further components. Further functional moieties maycomprise an amino acid component (AA) as defined herein or (AA)_(x),wherein (AA) is preferably an amino component as defined above. In theabove context, x is preferably an integer and may be selected from arange of about 1 to 100, preferably from a range of about 1 to 50, morepreferably 1 to 30, and even more preferably selected from a numbercomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-30, e.g.from a range of about 1 to 30, from a range of about 1 to 15, or from anumber comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15,or may be selected from a range formed by any two of the afore mentionedvalues. Most preferably, x is 1. Such an amino acid component (AA) or(AA)_(x) may be contained in every part of the inventive polymericcarrier according to formula (VIII) above and therefore may be attachedto all components of the inventive polymeric carrier according toformula (VII). It is particularly preferred that amino acid component(AA) or (AA)_(x) is present as a ligand or part of the repetitivecomponent [S—P²—S]_(n) within formula (VIII) of the inventive polymericcarrier.

In the context of the entire formula (VIII) of the polymeric carrier maybe preferably defined as follows:

L-P¹—S-[Cys-P²-Cys]_(n)-S—P³-L

wherein L, P¹, P², P³ and n are as defined herein, S is sulphur and eachCys provides for one —SH-moiety for the disulfide bond.

According to a particular embodiment, the polymeric carrier according toformula (VII) as defined above, may comprise at least one amino acidcomponent (AA) or (AA)_(x), as defined above. Such an amino acidcomponent (AA) or (AA)_(x) may be contained in every part of theinventive polymeric carrier according to formula (VIII) above andtherefore may be attached to all components of the polymeric carrieraccording to formula (VIII). It is particularly preferred that aminoacid component (AA) or (AA)_(x) is present as a ligand or part of therepetitive component [S—P²—S]_(n) within formula (VIII) of the polymericcarrier. The amino acid component (AA) or (AA)_(x) preferably containsor is flanked (e.g. terminally) by at least one —SH containing moiety,which allows introducing this component (AA) or (AA)_(x) via a disulfidebond into the polymeric carrier according to formula (VIII) as definedherein. Such a —SH-containing moiety may be any —SH containing moiety(or, of course, one sulphur of a disulfide bond), e.g. a cysteineresidue. In the specific case that the —SH containing moiety representsa cysteine, the amino acid component (AA)_(x) may also be read as-Cys-(AA)_(x)- or -Cys-(AA)_(x)-Cys- wherein Cys represents Cysteine andprovides for the necessary —SH-moiety for a disulfide bond. The —SHcontaining moiety may be also introduced into the amino acid component(AA)_(x) using any of modifications or reactions as shown above forcomponents P¹, P² or P³. In the specific case that the amino acidcomponent (AA)_(x) is linked to two components of the polymeric carrieraccording to formula (VIII) it is preferred that (AA) or (AA)_(x)contains at least two —SH-moieties, e.g. at least two Cysteines,preferably at its terminal ends. This is particularly preferred if (AA)or (AA)_(x) is part of the repetitive component [S—P²—S]_(n).Alternatively, the amino acid component (AA) or (AA)_(x) is introducedinto the polymeric carrier according to formula (VIII) as defined hereinvia any chemical possible addition reaction. Therefore the amino acidcomponent (AA) or (AA)_(x) contains at least one further functionalmoiety, which allows attaching same to a further component as definedherein, e.g. component P¹ or P³, P², L, or a further amino acidcomponent (AA) or (AA)_(x), etc. Such functional moieties may beselected from functionalities which allow the attachment of furthercomponents, e.g. functionalities as defined herein, e.g. by amideformation (e.g. carboxylic acids, sulphonic acids, amines, etc), byMichael addition (e.g maleinimide moieties, α,β unsatured carbonyls,etc), by click chemistry (e.g. azides or alkines), by alkene/alkinemethatesis (e.g. alkenes or alkines), imine or hydrozone formation(aldehydes or ketons, hydrazins, hydroxylamins, amines), complexationreactions (avidin, biotin, protein G) or components which allowS_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols,amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphoniumsalts) or other chemical moieties which can be utilized in theattachment of further components.

The amino acid component (AA) or (AA)_(x) in the polymeric carrier offormula (VIII) may also occur as a mixed repetitive amino acid component[(AA)_(x)]_(z), wherein the number of amino acid components (AA) or(AA)_(x) is further defined by integer z. In this context, z may beselected from a range of about 1 to 30, preferably from a range of about1 to 15, more preferably 1 to 10 or 1 to 5 and even more preferablyselected from a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15, or may be selected from a range formed by any two ofthe afore mentioned values.

According to a specific and particularly preferred alternative, theamino acid component (AA) or (AA)_(x), preferably written asS-(AA)_(x)-S or [S-(AA)_(x)-S] may be used to modify component P²,particularly the content of component S—P²—S in repetitive component[S—P²—S]_(n) of the polymeric carrier of formula (VIII) above. This maybe represented in the context of the entire polymeric carrier accordingto formula (VIII) e.g. by following formula (Villa):

L-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-L,

wherein x, S, L, AA, P¹, P² and P³ are preferably as defined herein. Informula (Villa) above, any of the single components [S—P²—S] and[S-(AA)_(x)-S] may occur in any order in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}. The numbers of single components[S—P²—S] and [S-(AA)_(x)-S] in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} are determined by integers a and b,wherein a+b=n. n is an integer and is defined as above for formula(VIII).

a is an integer, typically selected independent from integer b from arange of about 1 to 50, preferably from a range of about 1, 2 or 3 to30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or arange of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4,or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. arange of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range ofabout 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to11 or 7 to 10. Most preferably, a is in a range of about 1, 2, 3, 4, or5 to 10, more preferably in a range of about 1, 2, 3, or 4 to 9, in arange of about 1, 2, 3, or 4 to 8, or in a range of about 1, 2, or 3 to7.

b is an integer, typically selected independent from integer a from arange of about 0 to 50 or 1 to 50, preferably from a range of about 0,1, 2 or 3 to 30, more preferably from a range of about 0, 1, 2, 3, 4, or5 to 25, or a range of about 0, 1, 2, 3, 4, or 5 to 20, or a range ofabout 0, 1, 2, 3, 4, or 5 to 15, or a range of about 0, 1, 2, 3, 4, or 5to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or arange of about 6 to 11 or 7 to 10. Most preferably, b is in a range ofabout 1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2,3, or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range ofabout 1, 2, or 3 to 7.

In this context it is particularly preferred that the RNA, preferablymRNA of the inventive composition is complexed at least partially with acationic or polycationic compound and/or a polymeric carrier, preferablycationic proteins or peptides. In this context the disclosure of WO2010/037539 and WO 2012/113513 is incorporated herewith by reference.Partially means that only a part of the RNA is complexed with a cationiccompound and that the rest of the RNA is (comprised in the inventivecomposition) in uncomplexed form (“free”). Preferably the ratio ofcomplexed RNA to:free RNA (in the inventive composition) is selectedfrom a range of about 5:1 (w/w) to about 1:10 (w/w), more preferablyfrom a range of about 4:1 (w/w) to about 1:8 (w/w), even more preferablyfrom a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), andmost preferably the ratio of complexed RNA to free RNA in the inventivecomposition is selected from a ratio of about 1:1 (w/w).

The so called “(adjuvant) component”, which may be used to together withthe RNA, preferably mRNA in the inventive composition, is preferablyprepared according to a first step by complexing the at least one (m)RNAof the (adjuvant) component with a cationic or polycationic compoundand/or with a polymeric carrier, preferably as defined herein, in aspecific ratio to form a stable complex. In this context, it is highlypreferable, that no free cationic or polycationic compound or polymericcarrier or only a neglectably small amount thereof remains in the(adjuvant) component after complexing the (m)RNA. Accordingly, the ratioof the (m)RNA and the cationic or polycationic compound and/or thepolymeric carrier in the (adjuvant) component is typically selected in arange that the (m)RNA is entirely complexed and no free cationic orpolycationic compound or polymeric carrier or only a neglectably smallamount thereof remains in the composition. Preferably the ratio of the(adjuvant) component, i.e. the ratio of the (m)RNA to the cationic orpolycationic compound and/or the polymeric carrier, preferably asdefined herein, is selected from a range of about 6:1 (w/w) to about0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w),even more preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1(w/w) to about 2:1 (w/w). Alternatively, the ratio of the (m)RNA to thecationic or polycationic compound and/or the polymeric carrier,preferably as defined herein, in the (adjuvant) component, may also becalculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) ofthe entire complex. In the context of the present invention, anN/P-ratio is preferably in the range of about 0.1-10, preferably in arange of about 0.3-4 and most preferably in a range of about 0.5-2 or0.7-2 regarding the ratio of RNA:cationic or polycationic compoundand/or polymeric carrier, preferably as defined herein, in the complex,and most preferably in the range of about 0.7-1.5, preferably providedthe cationic or polycationic compound in the complex is a cationic orpolycationic cationic or polycationic protein or peptide and/or thepolymeric carrier is as defined herein. Such ratios, particularly weightand/or N/P ratios may also be applied to ratios of the at least one RNAas defined herein to a cationic or polycationic polymer or a polymericcarrier as defined herein used to complex the at least one RNA.

In this context, the N/P ratio is a measure of the ionic charge of thecationic (side chain) component of the cationic or polycationic compoundor. In particular, if the cationic properties of the cationic compoundare generated by nitrogens (e.g. of the amino acid side chains), the N/Pratio expresses the ratio of basic nitrogen atoms to phosphate residuesin the nucleotide backbone, considering that (side chain) nitrogen atomsin the cationic compound contribute to positive charges and phosphate ofthe phosphate backbone of the nucleic acid contribute to the negativecharge. The N/P-ratio is defined as the nitrogen/phosphate ratio(N/P-ratio) of the entire complex of nucleic acid and cationic orpolycationic compound. This is typically illustrative for thecontent/amount of cationic compounds and characteristic for thecontent/amount of nucleic acids bound or complexed. It may be calculatedon the basis that, for example, 1 μg RNA typically contains about 3 nmolphosphate residues, provided that RNA exhibits a statisticaldistribution of bases. Additionally, 1 nmol peptide typically containsabout x nmol nitrogen residues, dependent on the molecular weight andthe number of its (cationic) amino acids.

According to a particularly preferred embodiment the inventivecomposition comprises a polymeric carrier cargo complex comprising orconsisting of

-   -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components preferably as defined        above, more preferably according to formula VII, VIIa, VIIb or        VIII, and    -   b) as a cargo at least one nucleic acid molecule, preferably an        immunostimulating RNA, most preferably an RNA comprising an RNA        sequence according to SEQ ID NOs. 5, 394, or 10072, preferably        for use as a medicament, more preferably for use as an        immunostimulating agent or adjuvant, preferably for the        treatment of cancer or tumor diseases, wherein the polymeric        carrier cargo complex is preferably administered intratumorally.

In a preferred embodiment, the inventive RNA containing compositioncomprises a polymeric carrier cargo complex, comprising:

-   -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components, preferably as defined        above, more preferably according to formula VII, VIIa, VIIb or        VIII, and    -   a) b) as a cargo at least one first nucleic acid molecule,        preferably an immunostimulating RNA, most preferably an RNA        comprising an RNA sequence according to SEQ ID NOs. 5, 394, or        10072,

for use as an immunostimulating agent or as an adjuvant,

and at least one second nucleic acid molecule, preferably an RNA andmore preferably an mRNA encoding at least one protein or a peptide mostpreferably as disclosed above for coding RNA, and wherein the inventivecomposition is preferably administered intratumorally.

In a preferred embodiment, the invention relates to a polymeric carriercargo complex, comprising:

-   -   a) as a carrier a polymeric carrier formed by        disulfide-crosslinked cationic components, preferably as defined        above, more preferably according to formula VII, VIIa, VIIb or        VIII, and    -   a) b) as a cargo at least one first nucleic acid molecule,        preferably an immunostimulating RNA, most preferably an RNA        comprising an RNA sequence according to SEQ ID NOs. 5, 394, or        10072,

for use as an immunostimulating agent or as an adjuvant,

wherein the polymeric carrier cargo complex is administered incombination with at least one second nucleic acid molecule, preferablyan RNA and more preferably an mRNA encoding at least one protein or apeptide most preferably as disclosed above for coding RNA, and

wherein the polymeric carrier cargo complex and the second nucleic acidmolecule are preferably administered intratumorally.

Such preferred combinations of at least one first nucleic acid,preferably an immunostimulating RNA and at least one second nucleicacid, preferably an RNA, and more preferably an mRNA encoding at leastone protein or peptide are disclosed above in the context of“combinations of coding and non-coding RNA”.

As used herein, the term “first nucleic acid molecule” refers to anucleic molecule, which is used as a cargo in the polymeric carriercargo complex and is thus associated with the polymeric carrier. Theterm “second nucleic acid molecule”, as used herein, typically refers toa nucleic acid, which is not part of the polymeric carrier cargo complexand which encodes at least one peptide or protein.

In the context of the present invention immunostimulating agents oradjuvants are understood as compounds, which are preferably efficient ininducing an innate immune response, particularly in inducing theanti-viral cytokine IFN-alpha.

Adjuvants or immunostimulating agents usually act via their capabilityto induce an innate immune response. The innate immune system forms thedominant system of host defense in most organisms and comprises barrierssuch as humoral and chemical barriers including, e.g., inflammation, thecomplement system and cellular barriers. The innate immune system istypically based on a small number of receptors, called patternrecognition receptors. They recognize conserved molecular patterns thatdistinguish foreign organisms, like viruses, bacteria, fungi andparasites, from cells of the host. Such pathogen-associated molecularpatterns (PAMP) include viral nucleic acids, components of bacterial andfungal walls, flagellar proteins, and more. The first family of patternrecognition receptors (PAMP receptors) studied in detail was theToll-like receptor (TLR) family. TLRs are transmembrane proteins whichrecognize ligands of the extracellular milieu or of the lumen ofendosomes. Following ligand-binding they transduce the signal viacytoplasmic adaptor proteins which leads to triggering of a host-defenceresponse and entailing production of antimicrobial peptides,proinflammatory chemokines and cytokines, antiviral cytokines, etc. (seee.g. Meylan, E., J. Tschopp, et al. (2006), Nature 442(7098): 39-44).Further relevant components of the immune system include e.g. theendosomal TLRs, cytoplasmic receptors, Type I interferons andcytoplasmic receptors. Therefore, the immunostimulating agents oradjuvants are defined herein preferably as inducers of an innate immuneresponse, which activate pattern recognition receptors (PAMP receptors).Hereby, a cascade of signals is elicited, which e.g. may result in therelease of cytokines (e.g. IFN-alpha) supporting the innate immuneresponse. Accordingly, it is preferably a feature of animmunostimulating agent or adjuvant to bind to such receptors andactivate such PAMP receptors. Ideally, such as an agent or adjuvantadditionally supports the adaptive immune response by e.g. shifting theimmune response such that the preferred class of Th cells is activated.Depending on the disease or disorder to be treated a shift to aTh1-based immune response may be preferred or, in other cases, a shiftto a Th2 immune response may be preferred. Furthermore, adjuvants areusually defined as compounds that can increase and/or modulate theintrinsic immunogenicity of an antigen.

The term “immunostimulating agent” is typically understood not toinclude agents as e.g. antigens (of whatever chemical structure), whichelicit an adaptive/cytotoxic immune response, e.g. a “humoral” or“cellular” immune response, in other words elicit immune responses (andconfer immunity by themselves) which are characterized by a specificresponse to structural properties of an antigen recognized to be foreignby immune competent cells. Rather “immunostimulating agent” is typicallyunderstood to mean agents/compounds/complexes which do not trigger anyadaptive immune response by themselves, but which may exclusivelyenhance such an adaptive immune response in an unspecific way, by e.g.activating “PAMP” receptors and thereby triggering the release ofcytokines which support the actual adaptive immune response.Accordingly, any immunostimulation by agents (e.g. antigens) which evokean adaptive immune response by themselves (conferring immunity bythemselves directly or indirectly) is typically disclaimed by the phrase“immunostimulating agent”.

The term “adjuvant” is also understood not to comprise agents whichconfer immunity by themselves.

Accordingly, adjuvants do not by themselves confer immunity, but assistthe immune system in various ways to enhance the antigen-specific immuneresponse by e.g. promoting presentation of an antigen to the immunesystem. Hereby, an adjuvant may preferably e.g. modulate theantigen-specific immune response by e.g. shifting the dominatingTh2-based antigen specific response to a more Th1-based antigen specificresponse or vice versa. Accordingly, the terms “immunostimulating agent”and “adjuvant” in the context of the present invention are typicallyunderstood to mean agents, compounds or complexes which do not conferimmunity by themselves, but exclusively support the immune response inan unspecific way (in contrast to an antigen-specific immune response)by effects, which modulate the antigen-specific (adaptive cellularand/or humoral immune response) by unspecific measures, e.g. cytokineexpression/secretion, improved antigen presentation, shifting the natureof the arms of the immune response etc. Accordingly, any agents evokingby themselves immunity are typically disclaimed by the terms “adjuvant”or “immunostimulating agent”.

The use of the polymeric carrier cargo complex optionally in combinationwith a second nucleic acid molecule, preferably an RNA, allows provisionof a more efficient and/or safer medicament. Advantageously, thepolymeric carrier cargo complex is suited for in vivo delivery ofnucleic acids, in particular for compacting and stabilizing a nucleicacid for the purposes of nucleic acid transfection, such as exhibitingone or more reduced negative side effects of high-molecular weightpolymers as discussed above, such as poor biodegradability or hightoxicity, agglomeration, low transfection activity in vivo, etc. Thepolymeric carrier cargo complex also provides for improved nucleic acidtransfer in vivo, particularly via intratumoral routes, including serumstability, salt stability, efficiency of uptake, reduced complementactivation, nucleic acid release, etc. Such a polymeric carrier cargocomplex furthermore may support induction and maintenance of an adaptiveimmune response by initiating or boosting a parallel innate immuneresponse. It has been found that an improved adaptive immune responsecan further be obtained, in particular when the polymeric carrier cargocomplex is administered in combination with a second nucleic acidmolecule, preferably an RNA, encoding a protein or peptide, or when thepolymeric carrier cargo complex is co-formulated in a pharmaceuticalcomposition with a second nucleic acid molecule, preferably an RNA,encoding a protein or peptide, preferably an antigenic peptide orprotein. It has proven as particularly beneficial in this respect toadminister the inventive composition comprising the polymeric carriercargo complex optionally in combination with the second nucleic acidmolecule as defined herein via an intratumoral route. Additionally, thepolymeric carrier cargo complex may exhibit improved storage stability,particularly during lyophilisation.

In particular embodiments, the polymeric carrier cargo complex asdefined above enhances the immune response against a protein or peptide,which is encoded by a second nucleic acid molecule, preferably an RNA,more preferably an mRNA, that is administered in combination with thepolymeric carrier cargo complex, preferably via an intratumoral route ofadministration.

The polymeric carrier cargo complex and/or the second nucleic acidmolecule encoding a peptide or protein are preferably provided togetherwith a pharmaceutically acceptable carrier and/or vehicle. In thecontext of the present invention, a pharmaceutically acceptable carriertypically includes the liquid or non-liquid material, which is mixedwith the polymeric carrier cargo complex and/or the second nucleic acidmolecule. If the polymeric carrier cargo complex and/or the secondnucleic acid molecule are provided in liquid form, the carrier willtypically be pyrogen-free water; isotonic saline or buffered aqueoussolutions, e.g phosphate, citrate etc. buffered solutions. Ringer orRinger-Lactate solution is particularly preferred as a liquid basis.

The phrase “administered in combination” as used herein refers to asituation, where the polymeric carrier cargo complex is administered toa subject before, concomittantly or after the administration of thesecond nucleic acid molecule encoding a protein or peptide to the samesubject. Preferably, the time interval between the administration of thepolymeric carrier cargo complex and the at least one second nucleic acidmolecule, preferably an RNA, encoding a protein or peptide is less thanabout 48 hours, more preferably less than about 24 hours, 12 hours, 6hours, 4 hours, 2 hours, 1 hour, most preferably less than about 30minutes, 15 minutes or 5 minutes. In a particularly preferredembodiment, the phrase “administered in combination” refers toconcomitant administration of the polymeric carrier cargo complex andthe at least one second nucleic acid molecule, i.e. the simultaneousadministration of both components or the administration of bothcomponents within a time frame that typically comprises less than 5minutes. The phrase “administered in combination” does not only refer toa situation, where the pharmaceutical carrier cargo complex is inphysical contact with the at least one second nucleic acid molecule orformulated together with said second nucleic acid molecule. The phrase“administered in combination” as used herein comprises also the separateadministration of the polymeric carrier cargo complex and the secondnucleic acid molecule (e.g. by two separate injections), as long as thetime interval between the two administrations does not exceed theinterval as defined above. Alternatively, the polymeric carrier cargocomplex and the second nucleic acid molecule may be administered incombination by mixing the polymeric carrier cargo complex and the secondnucleic acid molecule prior to administration and administering themixture to a subject. When the polymeric carrier cargo complex isformulated together with the second nucleic acid molecule or when acomposition as defined herein is used, the polymeric carrier cargocomplex and the second nucleic acid molecule may further, independentlyfrom each other, administered in combination via any of theadministration routes as described herein.

The polymeric carrier cargo complex comprises as a cargo at least onenucleic acid molecule. In the context of the present invention, such anucleic acid molecule may be any suitable nucleic acid, selected e.g.from any (single-stranded or double-stranded) DNA, preferably, withoutbeing limited thereto, e.g. genomic DNA, single-stranded DNA molecules,double-stranded DNA molecules, coding DNA, DNA primers, DNA probes,immunostimulatory/immunostimulating DNA, a (short) DNA oligonucleotide((short) oligodesoxyribonucleotides), viral DNA, or may be selected e.g.from any PNA (peptide nucleic acid) or may be selected e.g. from any(single-stranded or double-stranded) RNA, preferably, without beinglimited thereto, a (short) RNA oligonucleotide ((short)oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), a viral RNA,replicons, an immunostimulatory/immunostimulating RNA, a smallinterfering RNA (siRNA), an antisense RNA, a micro RNA, a small nuclearRNA (snRNA), a small-hairpin (sh) RNA or riboswitches, ribozymes oraptamers; etc. The nucleic acid molecule of the polymeric carrier cargocomplex may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), amessenger RNA (mRNA), or a viral RNA (vRNA). Preferably, the nucleicacid molecule of the polymeric carrier cargo complex is an RNA. Morepreferably, the nucleic acid molecule of the polymeric carrier cargocomplex is a (linear) single-stranded RNA, even more preferably an mRNAor an immunostimulatory/immunostimulating RNA.

Furthermore, the nucleic acid of the polymeric carrier cargo complex maybe a single- or a double-stranded nucleic acid molecule or a partiallydouble-stranded or partially single stranded nucleic acid, which are atleast partially self complementary (both of these partiallydouble-stranded or partially single stranded nucleic acid molecules aretypically formed by a longer and a shorter single-stranded nucleic acidmolecule or by two single stranded nucleic acid molecules, which areabout equal in length, wherein one single-stranded nucleic acid moleculeis in part complementary to the other single-stranded nucleic acidmolecule and both thus form a double-stranded nucleic acid molecule inthis region, i.e. a partially double-stranded or partially singlestranded nucleic acid molecule. Preferably, the nucleic acid moleculemay be a single-stranded nucleic acid molecule. Furthermore, the nucleicacid molecule may be a circular or linear nucleic acid molecule,preferably a linear nucleic acid molecule.

According to one alternative, the nucleic acid molecule of the inventivepolymeric carrier cargo complex may be a coding nucleic acid, e.g. a DNAor RNA. Moreover, the polymeric carrier cargo complex may beadministered in combination with at least one second nucleic acidmolecule, which encodes a protein or a peptide.

According to one embodiment, the at least one first nucleic acidmolecule and the at least one second nucleic acid molecule are bothcoding nucleic acid molecules. Preferably, the at least one first andthe at least one second nucleic acid molecule each encode a differentpeptide or protein. In one embodiment, the first nucleic acid moleculehas a sequence, which is distinct from the sequence of the secondnucleic acid molecule, which is administered in combination with thepolymeric carrier cargo complex. Alternatively, the first nucleic acidmolecule and the second nucleic acid molecule may comprise the samesequence or be identical.

In the case of the at least one first nucleic acid molecule and/or ofthe second nucleic acid molecule, such a coding DNA or RNA may be anyDNA or RNA as defined herein. Preferably, such a coding DNA or RNA maybe a single- or a double-stranded DNA or RNA, more preferably asingle-stranded DNA or RNA, and/or a circular or linear DNA or RNA, morepreferably a linear DNA or RNA. Furthermore such a coding DNA or RNA maybe a genomic DNA, a viral RNA or DNA, a replicon, a plasmid DNA or anmRNA. Even more preferably, the coding DNA or RNA may be a (linear)single-stranded DNA or RNA. Most preferably, the nucleic acid moleculeaccording to the present invention may be a linear single-strandedmessenger RNA (mRNA). Such an mRNA may occur as a mono-, di-, or evenmulticistronic RNA, i.e. an RNA which carries the coding sequences ofone, two or more proteins or peptides. Such coding sequences in di-, oreven multicistronic mRNA may be separated by at least one IRES sequence,e.g. as defined herein.

In a preferred embodiment, the at least one second nucleic acid moleculeencodes a therapeutically active protein or an antigen as definedherein, preferably as disclosed in the context of “coding RNA”. In aparticularly preferred embodiment, the at least one second nucleic acidmolecule, which is administered in combination with the polymericcarrier cargo complex, encodes a peptide or a protein, which is capableof eliciting an immune response, preferably an adaptive immune response,after administration, especially intratumoral administration, to a host.Alternatively, the at least one second nucleic acid molecule encodes atleast one therapeutically active peptide or protein, preferably selectedfrom the group consisting of cytokines, chemokines, suicide geneproducts, immunogenic proteins or peptides, apoptosis inducers,angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenininhibitors, activators of the STING pathway, checkpoint modulators,innate immune activators, antibodies, dominant negative receptors anddecoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs),IDO pathway inhibitors, and proteins or peptides that bind inhibitors ofapoptosis.

In a particular embodiment, the first nucleic acid molecule of theherein defined polymeric carrier cargo complex and/or the second nucleicacid molecule administered in combination with the polymeric carriercargo complex may contain backbone modifications, sugar modifications orbase modifications. A backbone modification in connection with thepresent invention is a modification in which phosphates of the backboneof the nucleotides contained in the nucleic acid molecule of theinventive polymeric carrier cargo complex are chemically modified. Asugar modification in connection with the present invention is achemical modification of the sugar of the nucleotides of the firstnucleic acid molecule of the inventive polymeric carrier cargo complexand/or of the second nucleic acid molecule administered in combinationwith the polymeric carrier cargo complex. Furthermore, a basemodification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the nucleic acidmolecule of the inventive polymeric carrier cargo complex and/or of thesecond nucleic acid molecule administered in combination with thepolymeric carrier cargo complex. Such modifications are disclosed abovein the context of “RNA modifications”.

According to a further embodiment, the first nucleic acid molecule ofthe herein defined polymeric carrier cargo complex and/or the secondnucleic acid molecule administered in combination with the polymericcarrier cargo complex can contain a lipid modification. Such alipid-modified nucleic acid typically comprises a nucleic acid asdefined herein. Such a lipid-modified first nucleic acid molecule of thepolymeric carrier cargo complex or a lipid-modified second nucleic acidmolecule administered in combination with the polymeric carrier cargocomplex typically further comprises at least one linker covalentlylinked with that nucleic acid molecule, and at least one lipidcovalently linked with the respective linker. Alternatively, thelipid-modified nucleic acid molecule comprises at least one nucleic acidmolecule as defined herein and at least one (bifunctional) lipidcovalently linked (without a linker) with that nucleic acid molecule.According to a third alternative, the lipid-modified nucleic acidmolecule comprises a nucleic acid molecule as defined herein, at leastone linker covalently linked with that nucleic acid molecule, and atleast one lipid covalently linked with the respective linker, and alsoat least one (bifunctional) lipid covalently linked (without a linker)with that nucleic acid molecule.

According to a further preferred embodiment, the at least one RNA of theinventive composition is complexed with lipids to form one or moreliposomes, lipoplexes, or lipid nanoparticles. Therefore, in oneembodiment, the inventive composition comprises liposomes, lipoplexes,and/or lipid nanoparticles comprising the at least one RNA.

Lipid-based formulations have been increasingly recognized as one of themost promising delivery systems for RNA due to their biocompatibilityand their ease of large-scale production. Cationic lipids have beenwidely studied as synthetic materials for delivery of RNA. After mixingtogether, nucleic acids are condensed by cationic lipids to formlipid/nucleic acid complexes known as lipoplexes. These lipid complexesare able to protect genetic material from the action of nucleases anddeliver it into cells by interacting with the negatively charged cellmembrane. Lipoplexes can be prepared by directly mixing positivelycharged lipids at physiological pH with negatively charged nucleicacids.

Conventional liposomes consist of a lipid bilayer that can be composedof cationic, anionic, or neutral (phospho)lipids and cholesterol, whichencloses an aqueous core. Both the lipid bilayer and the aqueous spacecan incorporate hydrophobic or hydrophilic compounds, respectively.Liposome characteristics and behaviour in vivo can be modified byaddition of a hydrophilic polymer coating, e.g. polyethylene glycol(PEG), to the liposome surface to confer steric stabilization.Furthermore, liposomes can be used for specific targeting by attachingligands (e.g., antibodies, peptides, and carbohydrates) to its surfaceor to the terminal end of the attached PEG chains (Front Pharmacol. 2015Dec. 1; 6:286).

Liposomes are colloidal lipid-based and surfactant-based deliverysystems composed of a phospholipid bilayer surrounding an aqueouscompartment. They may present as spherical vesicles and can range insize from 20 nm to a few microns. Cationic lipid-based liposomes areable to complex with negatively charged nucleic acids via electrostaticinteractions, resulting in complexes that offer biocompatibility, lowtoxicity, and the possibility of the large-scale production required forin vivo clinical applications. Liposomes can fuse with the plasmamembrane for uptake; once inside the cell, the liposomes are processedvia the endocytic pathway and the genetic material is then released fromthe endosome/carrier into the cytoplasm. Liposomes have long beenperceived as drug delivery vehicles because of their superiorbiocompatibility, given that liposomes are basically analogs ofbiological membranes, and can be prepared from both natural andsynthetic phospholipids (Int J Nanomedicine. 2014; 9: 1833-1843).

Cationic liposomes have been traditionally the most commonly usednon-viral delivery systems for oligonucleotides, including plasmid DNA,antisense oligos, and siRNA/small hairpin RNA-shRNA). Cationic lipids,such as DOTAP, (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate)can form complexes or lipoplexes with negatively charged nucleic acidsto form nanoparticles by electrostatic interaction, providing high invitro transfection efficiency. Furthermore, neutral lipid-basednanoliposomes for RNA delivery as e.g. neutral1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomeswere developed. (Adv Drug Deliv Rev. 2014 February; 66: 110-116.).

Therefore, in one embodiment the at least one RNA of the inventivecomposition is complexed with cationic lipids and/or neutral lipids andthereby forms liposomes, lipid nanoparticles, lipoplexes or neutrallipid-based nanoliposomes.

Preferred cationic or polycationic compounds, which can be used astransfection or complexation agent may include cationic polysaccharides,for example chitosan, polybrene, cationic polymers, e.g.polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

Additional Pharmaceutically Active Compounds:

Furthermore the inventive composition may comprise at least oneadditional pharmaceutically active component/compound. Alternatively orin addition to that, the at least one additional pharmaceutically activecomponent/compound may be co-administered concomitant to the compositionaccording to the invention. Therefore, the at least one additionalpharmaceutically active component/compound may be administered incombination with the at least one RNA of the inventive composition orwith the RNA containing composition according to the invention.

The phrases “administered in combination”, co-administration or“concomitant administration” as used herein refers to a situation, wherethe inventive composition or an ingredient thereof is administered to asubject before, concomittantly or after the administration of a furtherpharmaceutically active component to the same subject. The time intervalbetween the administration of the inventive composition or an ingredientthereof and the at least one second pharmaceutically active componentdepends on the nature and biological effect of the particularpharmaceutically active component and can be determined by a physician.Preferably the time interval is less than about 48 hours, morepreferably less than about 24 hours, 12 hours, 6 hours, 4 hours, 2hours, 1 hour, most preferably less than about 30 minutes, 15 minutes or5 minutes. In a particularly preferred embodiment, the phrase“administered in combination” refers to concomitant administration ofthe inventive composition or an ingredient thereof and the at least onesecond pharmaceutically active component, i.e. the simultaneousadministration of both compounds or the administration of both compoundswithin a time frame that typically comprises less than 5 minutes. Thephrase “administered in combination” does not only refer to a situation,where the inventive composition or an ingredient thereof is in physicalcontact with the at least one second pharmaceutically active componentor formulated together with said second pharmaceutically activecomponent. The phrase “administered in combination” as used hereincomprises also the separate administration of the inventive compositionor an ingredient thereof and the second pharmaceutically activecomponent (e.g. by two separate injections). Alternatively, theinventive composition or an ingredient thereof and the secondpharmaceutically active component may be administered in combination bymixing the inventive composition or an ingredient thereof and the secondpharmaceutically active component prior to administration andadministering the mixture to a subject. When the inventive compositionor an ingredient thereof is formulated together with the secondpharmaceutically active component or when a composition as definedherein is used, the inventive composition or an ingredient thereof andthe second pharmaceutically active component may further, independentlyfrom each other, administered in combination via any of theadministration routes as described herein.

A pharmaceutically active component/compound in this connection is acompound that has a therapeutic effect to heal, ameliorate or prevent aparticular indication or disease, namely a tumor or cancer disease. Suchcompounds include, without implying any limitation, peptides orproteins, preferably as defined herein, nucleic acids, preferably asdefined herein, (therapeutically active) low molecular weight organic orinorganic compounds (molecular weight less than 5000, preferably lessthan 1000), sugars, antigens or antibodies, preferably as definedherein, therapeutic agents already known in the prior art, antigeniccells, antigenic cellular fragments, cellular fractions, cell wallcomponents (e.g. polysaccharides), modified, attenuated or de-activated(e.g. chemically or by irradiation) pathogens (virus, bacteria etc.),adjuvants, etc.

In a preferred embodiment, the inventive composition additionallycomprises at least one further pharmaceutically activecomponent/compound, wherein the at least one additional pharmaceuticallyactive component is selected from cytokines, chemokines, suicide geneproducts, immunogenic proteins or peptides, apoptosis inducers,angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenininhibitors, activators of the STING pathway, checkpoint modulators,innate immune activators, antibodies, dominant negative receptors anddecoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs),IDO pathway inhibitors, proteins or peptides that bind inhibitors ofapoptosis, anti-bacterial agents, anti-viral agents, adjuvants,chemotherapeutic agents and kinase inhibitors.

Alternatively, or in addition to that, the at least one additionalpharmaceutically active component may be co-administered concomitant tothe at least one RNA of the RNA containing composition or the inventivecomposition or may be used in combination with the at least one RNA ofthe RNA containing composition or the inventive composition.

In this context protein-based cytokines, chemokines, suicide geneproducts, immunogenic proteins or peptides, apoptosis inducers,angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenininhibitors, activators of the STING pathway, checkpoint modulators,innate immune activators, antibodies, dominant negative receptors anddecoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs),IDO pathway inhibitors, and proteins or peptides that bind inhibitors ofapoptosis or fragments and variants thereof as disclosed above in thecontext of “coding RNA” may be used as additional pharmaceuticallyactive component. Alternatively, nucleic acids encoding these proteinsor fragments or variants thereof may be used as additionalpharmaceutically active component.

1. Cytokines:

In this context protein-based cytokines, or fragments and variantsthereof as disclosed above in the context of “coding RNA” may be used asadditional pharmaceutically active component. Alternatively, nucleicacids encoding these proteins or fragments or variants thereof may beused as additional pharmaceutically active component.

Preferably the cytokine is an interleukin (IL). One or more interleukinsmay be chosen e.g. from the following list: IL-1α, IL-1β, IL-1ra(antagonist), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10;IL-11, IL-12, IL-13, IL14, IL-15, IL-16, IL-17A, IL-17B, EL-17C, IL-17D,IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, IL-27, IL-28A/B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35.Moreover the cytokine may be one or more cytokines chosen from the TNFfamily, e.g. chosen from the following list: TNF, especially TNFα, LTα,LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L (CD154), FASL,CD27L, CD30L, 4-1BBL, TRAIL, RANK ligand. Further examples of preferredcytokines may be chosen from the following list: FLT3 ligand, G-CSF,GM-CSF, IFNα/β/ω, IFNγ, LIF, M-CSF, MIF, OSM, Stem Cell Factor, TGFβ1,TGFβ2, TGFβ3, TSLP ligand.

Particularly preferred are cytokines chosen from the following list:IL-12, IL-15, IL-2, IFNγ, TNFα, IL-18, IFNα, IL-1β, IL-32, IL-7, IL-21,IL-8, GM-CSF.

In this context particularly preferred are cytokines as disclosed inTable 1 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072, and is combined with at least onecytokine as defined above, preferably IL-2, IL-12, CD40L or IL-15 or afragment or variant thereof.

1. Chemokines

In this context protein-based chemokines, or fragments and variantsthereof as disclosed above in the context of “coding RNA” may be used asadditional pharmaceutically active component. Alternatively, nucleicacids encoding these proteins or fragments or variants thereof may beused as additional pharmaceutically active component.

Preferred chemokines may be chosen from the following list: CXCL1,CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11,CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1, CCL2, CCL3, CCL4, CCL5,CCL6, CCL7, CCL8, CCL9/10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16,CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26,CCL27, CCL28, XCL1, XCL2, CX3CL1.

In this context particularly preferred are chemokines as disclosed inTable 2 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least onechemokine as defined above or a fragment or variant thereof.

2. Suicide Enzymes

In this context protein-based suicide enzymes, or fragments and variantsthereof as disclosed above in the context of “coding RNA” may be used asadditional pharmaceutically active component. Alternatively, nucleicacids encoding these proteins or fragments or variants thereof may beused as additional pharmaceutically active component.

The suicide enzyme is preferably a nucleotide metabolizing enzyme.Preferably the suicide enzyme is used in combination with a prodrugwhich is a substrate of the suicide enzyme, and which is converted to acytotoxic compound by the suicide enzyme. One or more preferred suicideenzymes may be chosen from the following list: thymidine kinase,preferably a viral thymidine kinase, more preferrably Herpes simplexvirus thymidine kinase, Varicella zoster thymidine kinase; a plantthymidine kinase, preferably a tomato thymidine kinase; cytosinedeaminase, preferably bacterial cytosine deaminase or Yeast cytosinedeaminase; deoxynucleoside kinase, preferably Drosophila melanogasterdeoxynucleoside kinase; deoxycytidine kinase, preferably a mammaliandeoxycytidine kinase, purine nucleoside phosphorylase, preferably abacterial purine nucleoside phosphorylase.

In this context particularly preferred are suicide enzymes (suicide geneproducts) as disclosed in Table 3 and 4 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least onesuicide enzyme as defined above or a fragment or variant thereof.

3. Immunogenic Proteins or Peptides

In this context protein-based immunogenic proteins or peptides, orfragments and variants thereof as disclosed above in the context of“coding RNA” may be used as additional pharmaceutically activecomponent. Alternatively, nucleic acids encoding these proteins orpeptides or fragments or variants thereof may be used as additionalpharmaceutically active component.

The immunogenic protein or peptide is preferably a pathogenic antigen toutilize preexisting immunity against such antigens for treatment oftumor and/or cancer diseases. The memory immune response is triggeredand the immune system is strengthened for attacking tumor cells.

Preferred examples of immunogenic proteins or peptides for thisembodiment of the invention are proteins or peptides of widespreadpathogens, i.e. pathogens with which every organism, in particularmammals, preferably humans, has a high probability of being infected atleast once in his/her lifetime. These include, for example, anystructural or non-structural protein or peptide of:

-   -   influenza virus type A or B or any other orthomyxovirus        (influenza type C),    -   picornaviruses, such as rhinovirus or hepatitis A virus,    -   togaviruses, such as alphavirus or rubivirus, e.g. Sindbis,        Semliki-Forest or rubeolavirus (measles virus),    -   rubella virus (German measles virus),    -   coronaviruses, in particular subtypes HCV-229E or HCV-0C43,    -   rhabdoviruses, such as rabies virus,    -   paramyxoviruses, such as mumps virus,    -   reoviruses, such as group A, B or C rotavirus,    -   hepadnaviruses, such as hepatitis B virus,    -   papoviruses, such as human papillomaviruses (HPV) of any        serotype, especially from 1 to 75,    -   adenoviruses, in particular type 1 to 47,    -   herpesviruses, such as Herpes simplex virus 1, 2 or 3,    -   cytomegalovirus (CMV), preferably CMVpp65,    -   Epstein Barr virus (EBV),    -   vaccinia viruses and    -   the bacterium Chiamydophila pneumoniae (Chlamydia pneumoniae).

Further examples of preferred immunogenic proteins or peptides areproteins or peptides of pathogens which only seldom infect an organism.These proteins or peptide include, for example, any structural ornon-structural protein or peptide of:

-   -   Flaviviruses, such as dengue virus type 1 to 4, yellow fever        virus, West Nile virus, Japanese encephalitis virus    -   hepatitis C virus,    -   caliciviruses,    -   filoviruses, such as Ebola virus,    -   bornaviruses,    -   bunyaviruses, such as Rift Valley fever virus,    -   arenaviruses, such as LCMV (lymphocytic choriomeningitis virus)        or hemorrhagic fever viruses,    -   retroviruses, such as HIV and    -   parvoviruses.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least oneimmunogenic protein or peptide as defined above, preferably influenzanucleoprotein (NP) or a fragment or variant thereof.

4. Apoptosis Inducers:

In this context protein-based apoptosis inducers, or fragments andvariants thereof as disclosed above in the context of “coding RNA” maybe used as additional pharmaceutically active component. Alternatively,nucleic acids encoding these proteins or peptides or fragments orvariants thereof may be used as additional pharmaceutically activecomponent.

Preferably, an apoptosis inducer is chosen from the group consisting ofthe Bcl-2 family and tumor suppressor protein p53 and ligands oftransmembrane death receptors, especially the TNF (tumor necrosisfactor) receptor gene superfamily, pro-apoptic receptor agonists andBeclin-1.

A particularly preferred apoptosis inducer in the context of the presentinvention is Beclin-1 (derived from the BECN1 gene).

Further preferred examples of apoptosis inducers may be chosen from thefollowing list: Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, Blk, Cytochrome c,Caspases, especially Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase9, Death domain, especially of Fas, preferably FasL, TNFα, Apo2L/TRAIL,agonist of DR4 and/or DR5, Apo3L, DR4 agonistic antibody, DR5 agonisticantibody, protein kinase R (PKR) (preferably constitutive active PKR),Granzyme B.

In this context particularly preferred are apoptosis inducers asdisclosed in Table 5 and 6 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least oneapoptosis inducer as defined above, or a fragment or variant thereof.

5. Angiogenesis Inhibitors

In this context protein-based angiogenesis inducers, or fragments andvariants thereof as disclosed above in the context of “coding RNA” maybe used as additional pharmaceutically active component. Alternatively,nucleic acids encoding these proteins or peptides or fragments orvariants thereof may be used as additional pharmaceutically activecomponent.

Preferred examples of angiogenesis inhibitors according to the inventionmay be chosen from the following list: interferon alpha (IFN-α),(interferon beta) IFN-β, interferon gamma (IFN-γ), CXCL9, CXCL10,interleukin 12 (IL-12), platelet factor 4 (PF-4), tumor necrosis factoralpha (TNF-α), soluble fms-like tyrosine kinase 1 (sFLT-1), Fetal LiverKinase 1 (FLK-1), Angiostatin, Endostatin, Vasostatin, Canstatin,Tumstatin, 16 kD prolacin fragment, tissue inhibitor ofmetalloproteinases 1 (TIMP-1), tissue inhibitor of metalloproteinases 2(TIMP-2), tissue inhibitor of metalloproteinases 3 (TIMP-3),thrombospondin 1 (TSP-1), thrombospondin 2 (TSP-2), Maspin, PEX, solubleTyrosine-protein kinase receptor 1 (sTie1), soluble Angiopoietin-1receptor 2 (sTie2), Angiopoietin-1, Angiopoietin-2, Antivascularendothelial growth factor receptor 2 (VEGFR2) antibody (e.g. Alacizumab,Ramucirumab), Anti-vascular endothelial growth factor (VEGF) antibody(e.g. Brolucizumab, Ranibizumab, Bevacizumab), and Anti-vascularendothelial growth factor receptor 1 (VEGFR1) antibody (e.g. Icrucumab).

In this context particularly preferred are angiogenesis inhibitors asdisclosed in Table 7 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least oneangiogenesis inhibitor as defined above, or a fragment or variantthereof.

6. Heat Shock Proteins:

In this context protein-based heat-shock proteins, or fragments andvariants thereof as disclosed above in the context of “coding RNA” maybe used as additional pharmaceutically active component. Alternatively,nucleic acids encoding these proteins or peptides or fragments orvariants thereof may be used as additional pharmaceutically activecomponent.

Preferably, the heat shock protein may be chosen from the followinglist: HSP27, HSP47 (serpin H1), HSP60, HSP70, HSC70, GRP78 (BiP), HSP90,HSP110, GRP94 (gp96), GRP170 (ORP150), PDI/PDIA, CRT/CALR.

In this context particularly preferred are heat shock proteins asdisclosed in Table 8 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least one heatshock protein as defined above, or a fragment or variant thereof.

7. Tumour Antigens:

In this context protein-based tumor antigens, or fragments and variantsthereof as disclosed above in the context of “coding RNA” may be used asadditional pharmaceutically active component. Alternatively, nucleicacids encoding these proteins or peptides or fragments or variantsthereof may be used as additional pharmaceutically active component.

In this context particularly preferred are tumor antigens as disclosedin Table 9 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least one tumorantigen as defined above, or a fragment or variant thereof.

8. β-Catenin Inhibitors:

In this context protein-based β-catenin inhibitors, or fragments andvariants thereof as disclosed above in the context of “coding RNA” maybe used as additional pharmaceutically active component. Alternatively,nucleic acids encoding these proteins or peptides or fragments orvariants thereof may be used as additional pharmaceutically activecomponent.

Particular preferred β-catenin inhibitors according to the presentinvention comprise TAT-NLS-BLBD-6, axin-1, TCF-4, GSK-3b, DKK-1, Dvl-1derivatives or fragments thereof.

Chemical β-Catenin Inhibitors:

According to the present invention, the at least one additional activepharmaceutical ingredient which may be contained in the inventivecomposition, and/or which may be co-administered, or which may becombined with the inventive composition may be a chemical β-catenininhibitors. Chemical β-catenin inhibitors are known in the art that maybe administered according to the present invention. Preferably thechemical β-catenin inhibitor is chosen from the following list:PKF118-310, CGP049090, PKF115-584, PKF222-815, PKF118-744, ICG001,CCT036477, XAV939, acyl hydrazones (HQBA), molecules with2,3,6-trisubstituted pyrido[2,3,-b]pyrazine core skeletons, carnosicacid, CCT031374, iCRT-3,5,14, NC043, Ibuprofin, aspirin.

The following table 13 summarizes examples of small molecular inhibitorsof β-catenin signaling which are particularly preferred in this context.

TABLE 13 β-catenin inhibitors Inhibitor Target Reference PKF118-310,CGP049090, PKF115- beta-catenin—TCF Lepourcelet et al., 2004. 584,PKF222-815 and PKF118-744 interaction Cancer Cell 5:91-102 ICG001beta-catenin—CBP Emami et al., 2004. Proc Natl interaction Acad Sci USA101:12682-7 CCT036477 beta-catenin—TCF Ewan et al., 2010. Cancerinteraction Res. 70:5963-73 XAV939 Tankyrase Huang et al., 2009. Nature461:614-20 acyl hydrazones (HQBA) Iron chelators Song et al., 2011.Cancer Res. 71:7628-39; Coombs et al., 2012. Oncogene 31:213-25molecules with 2,3,6-trisubstituted beta-catenin Gong et al., 2011.Bioorg pyrido[2,3,-b] pyrazine core Med Chem. 19:5639-47 skeletonscarnosic acid beta-catenin/BCL9 de la Roche et al., Nat Commun. 3:680CCT031374 beta-catenin Thorne et al., 2010. Nat Chem Biol. 6:829-36iCRT-3,5,14, NC043 beta-catenin—TCF Wang et al., 2011. Cell Res.interaction 21:730-40; Gonsalves et al., 2011. Proc Natl Acad Sci USA108:5954-63 Ibuprofin, aspirin Cox2 Inhibitors Greenspan et al., 2011.Cancer Prev Res. 4:161-71

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least oneβ-catenin inhibitor as defined above, or a fragment or variant thereof.

9. Activators of the STING Pathway

In this context protein-based activators of the STING pathway, orfragments and variants thereof as disclosed above in the context of“coding RNA” may be used as additional pharmaceutically activecomponent. Alternatively, nucleic acids encoding these proteins orpeptides or fragments or variants thereof may be used as additionalpharmaceutically active component. Preferably, the at least oneactivator (stimulator) of the STING pathway is chosen from an activatingprotein or a constitutively active protein of the STING pathway,preferably DDX41, STING, cGAS, IRF3, TBK1 or STAT6 or a fragment orvariant thereof.

Chemical STING-Pathway Activators:

In a further preferred embodiment the optional additionalpharmaceutically active component may be selected from chemicalactivators of the STING pathway which are preferably selected fromcyclic dinucleotides and xanthenone analogs.

Table 14 shows examples of chemical STING agonists. Further examples ofSTING agonists are disclosed in WO2014189805.

TABLE 14 Activators of STING pathway Class of STING activator examplescyclic dinucleotides 3′3′-cGAMP, 2′3′-cGAMP, 2′2′-cGAMP, c-di-APM,c-di-GMP, c-di-IMP, c-di-UMP xanthenone analogs DMXAA,5,6-dimethylxanthenone-4-acetic acid

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least one STINGpathway activator as defined above, or a fragment or variant thereof.

10. Checkpoint Modulators

In this context protein-based checkpoint modulators, or fragments andvariants thereof as disclosed above in the context of “coding RNA” maybe used as additional pharmaceutically active component. Alternatively,nucleic acids encoding these proteins or peptides or fragments orvariants thereof may be used as additional pharmaceutically activecomponent.

In preferred embodiments of the present invention the checkpointmodulator is a modulator of B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2,B7-H3, B7-H4, B7-H6, B7-H7/HHLA2, BTLA, CD28, CD28H/IGPR-1, CTLA-4,ICOS, PD-1, PD-L2/B7-DC, PDCD6, VISTA/B7-H5/PD-1H, BTN1A1/Butyrophilin,BTN2A1, BTN2A2/Butyrophilin 2A2, BTN3A1/2, BTN3A2, BTN3A3,BTNL2/Butyrophilin-like 2, BTNL3, BTNL4, BTNL6, BTNL8, BTNL9, BTNL10,CD277/BTN3A1, LAIR1, LAIR2, CD96, CD155/PVR, CRTAM, DNAM-1/CD226,Nectin-2/CD112, Nectin-3, TIGIT, LILRA3/CD85e, LILRA4/CD85g/ILT7,LILRB1/CD85j/ILT2, LILRB2/CD85d/ILT4, LILRB3/CD85a/ILT5,LILRB4/CD85k/ILT3, 4-1BB/TNFRSF9/CD137, 4-1BB Ligand/TNFSF9,BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7,CD30/TNFRSF8, CD30 Ligand/TNFSF8, CD40/TNFRSF5, CD40 Ligand/TNFSF5,DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14,LIGHT/TNFSF14, Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha,TNF RII/TNFRSF1B, 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9,CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7,NTB-A/SLAMF6, SLAM/CD150, TIM-1/KIM-1/HAVCR, TIM-3, TIM-4, CD7, CD96,CD160, CD200, CD300a/LMIR1, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26,EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3,TIM-1/KIM-1/HAVCR, TIM-4, TSLP R, or any combinations thereof.

Preferably, the checkpoint modulator is selected from agonisticantibodies, antagonistic antibodies, ligands, dominant negativereceptors and decoy receptors or combinations thereof.

Preferably, the agonistic antibody is chosen from the following list:anti-4-1BB, anti-OX40, anti-GITR, anti-CD28, anti-CD27,anti-CD-40anti-ICOS, anti-TNFRSF25, and anti-LIGHT.

Preferably, the antagonistic antibody is chosen from the list ofanti-CTLA4, anti-PD1, anti-PD-L1, anti-Vista, anti-Tim-3, anti-LAG-3,and anti-BTLA.

Particularly preferred are the anti-CTLA-4 antibodies ipilimumab(Yervoy®), tremelimumab, and AGEN-1884.

Particularly preferred are the anti-PD1 antibodies Nivolumab(MDX-1106/BMS-936558/ONO-4538), (Brahmer et al., 2010. J Clin Oncol.28(19):3167-75; PMID: 20516446); Pidilizumab (CT-011), (Berger et al.,2008. Clin Cancer Res. 14(10):3044-51; PMID: 18483370); Pembrolizumab(MK-3475, SCH 900475); AMP-224, and MED10680 (AMP-514).

Particularly preferred are the anti-PD-L1 antibodies MDX-1105/BMS-936559(Brahmer et al. 2012. N Engl J Med. 366(26):2455-65; PMID: 22658128);atezolizumab (MPDL3280A/RG7446); durvalumab (MED14736); and avelumab(MSB0010718).

According to the present invention checkpoint modulators according toTable 15 are particularly preferred:

TABLE 15 Antibodies directed against immune checkpoint proteins NameTarget Urelumab 4-1BB/CD137 PF-05082566 4-1BB/CD137 8H9 B7-H3Enoblituzumab B7-H3 Ipilimumab CD152/CTLA-4 Ticilimumab (=tremelimumab)CD152/CTLA-4 Tremelimumab CD152/CTLA-4 Varlilumab CD27 Teneliximab CD40Vorsetuzumab mafodotin CD70 Lirilumab KIR2D GSK-3174998 OX40 MEDI-6469OX40 MEDI-6383 OX40 MEDI-0562 OX40 PF-04518600 OX40 RG-7888 OX40PF-06801591 PD-1 BGBA-317 PD-1 MEDI-0680 PD-1 MK-3475 PD-1 NivolumabPD-1 PDR-001 PD-1 Pembrolizumab PD-1 Pidilizumab PD-1 REGN-2810 PD-1SHR-1210 PD-1 TSR-042 PD-1 MDX-1106 PD-1 Merck 3745 PD-1 CT-011 PD-1MEDI-0680 PD-1 PDR001 PD-1 REGN2810 PD-1 BGB-108 PD-1 BGB-A317 PD-1AMP-224 PD-1 Atezolizumab PD-L1 (CD274) Avelumab PD-L1 (CD274)BMS-936559 PD-L1 (CD274) Durvalumab PD-L1 (CD274) MEDI-4736 PD-L1(CD274) MPDL33280A PD-L1 (CD274) YW243.55.570 PD-L1 (CD274) MDX-1105PD-L1 (CD274) MSB0010718C PD-L1 (CD274)

In a further preferred embodiment the checkpoint modulator is a decoyreceptor (e.g. a soluble receptor). Preferably, the decoy receptor is asoluble PD1 receptor. In a particularly preferred embodiment the RNAsequence encoding a soluble PD1 receptor comprises an RNA sequenceaccording to SEQ ID NO: 389

In a further preferred embodiment the checkpoint modulator is a ligandof an immune checkpoint protein. Preferably, the ligand is CD40 Ligand(CD40L).

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least onecheckpoint modulator as defined above, preferably selected from ananti-CTLA4 antibody, an anti-PD1 antibody, an anti PD-L1 antibody, aCD40 ligand, or a soluble PD1 receptor, or a fragment or variantthereof.

11. Innate Immune Activators

In this context protein-based innate immune activators or fragments andvariants thereof as disclosed above in the context of “coding RNA” maybe used as additional pharmaceutically active component. Alternatively,nucleic acids encoding these proteins or peptides or fragments orvariants thereof may be used as additional pharmaceutically activecomponent.

In this context innate immune activators may be selected from mammalian,in particular human adjuvant proteins, which typically comprise anyhuman protein or peptide, which is capable of eliciting an innate immuneresponse (in a mammal), e.g. as a reaction of the binding of anexogenous TLR ligand to a TLR. More preferably, human adjuvant proteinsare selected from the group consisting of proteins which are componentsand ligands of the signalling networks of the pattern recognitionreceptors including TLR, NLR and RLH, including TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4,NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7,NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14, I IPAF, NAIP,CIITA, RIG-I, MDA5 and LGP2, the signal transducers of TLR signalingincluding adaptor proteins including e.g. Trif and Cardif; components ofthe Small-GTPases signalling (RhoA, Ras, Rac1, Cdc42, Rab etc.),components of the PIP signalling (PI3K, Src-Kinases, etc.), componentsof the MyD88-dependent signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP,TRAF6 etc.), components of the MyD88-independent signalling (TICAM1,TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinasesincluding e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2,GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK, p38MAPK, TAK1, IKK,and TAK1; the activated transcription factors including e.g. NF-κB,c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7.

Mammalian, in particular human adjuvant proteins may furthermore beselected from the group consisting of heat shock proteins, such asHSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIIIrepeat extra domain A of fibronectin; or components of the complementsystem including C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b,C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR,C1INH, C4bp, MCP, DAF, H, I, P and CD59, or induced target genesincluding e.g. Beta-Defensin, cell surface proteins; or human adjuvantproteins including trif, flt-3 ligand, Gp96 or fibronectin, etc., or anyspecies homolog of any of the above human adjuvant proteins. FurthermoreHGMB1 may be used as adjuvant protein.

Mammalian, in particular human adjuvant proteins may furthermorecomprise cytokines which induce or enhance an innate immune response,including IL-1 alpha, IL1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12,IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha,IFNbeta, IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8,IP-10, MCP-1, MIP-1alpha, RANTES, Eotaxin, CCL21; cytokines which arereleased from macrophages, including IL-1, IL-6, IL-8, IL-12 andTNF-alpha; as well as IL-1R1 and IL-1 alpha.

Therefore in this context it particularly preferred that the at leastinnate immune activator, is preferably an adjuvant protein, morepreferably a human adjuvant protein, or a fragment or variant thereof.

In this context it is particularly preferred that I constitutive activevariant of an adjuvant protein is used as innate immune activator,preferably a constitutive active variant of RIG-1 (ΔRIGI).

In another preferred embodiment the at least one innate immune activatoris HGMB1, or a fragment or variant thereof.

In this context particularly preferred are innate immune activators asdisclosed in Table 11 above.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least one innateimmune activator as defined above, or a fragment or variant thereof.

12. Antibodies, Decoy Receptors and Dominant Negative Receptors

In this context protein-based antibodies, decoy receptors, or dominantnegative receptors or fragments and variants thereof as disclosed abovein the context of “coding RNA” may be used as additionalpharmaceutically active component. Alternatively, nucleic acids encodingthese proteins or peptides or fragments or variants thereof may be usedas additional pharmaceutically active component.

According to the present invention antibodies according to Table 16 areparticularly preferred:

TABLE 16 Antibodies directed against proteins accociated with tumor orcancer development Name Target 3F8 GD2 Abagovomab CA-125 imitationAbciximab Platelet glycoprotein GPIIb/IIIa Adecatumumab EpCAM (CD326)Afutuzumab CD20 Alacizumab pegol VEGFR2 Alemtuzumab CD52 Altumomabpentetate CEA Amatuximab mesothelin Anatumomab mafenatox 5T4 Anetumabravtansine mesothelin Apolizumab HLA-DR beta apomab TRAIL-R2 (CD262)Arcitumomab CEA Ascrinvacumab ACVRL1 Bavituximab phosphatidylserineBectumomab CD22 Belimumab BAFF Besilesomab CEA Bevacizumab VEGF-ABivatuzumab mertansine CD44v6 Blinatumomab CD19 × CD3 Brentuximabvedotin CD30 (TNFRSF8) Brontictuzumab NOTCH1 canakinumab IL-1βCantuzumab mertansine CanAg Cantuzumab ravtansine MUC1 (CD227) Capromabpendetide PSMA Carlumab MCP-1 Catumaxomab EpCAM × CD3 cBR-doxorubicinimmunoconjugate CD174 (Lewis Y) Cetuximab EGFR (HER1/ERBB1) Citatuzumabbogatox EpCAM Cixutumumab IGF-1R Clivatuzumab tetraxetan MUC1 (CD227)Codrituzumab glypican 3 Coltuximab ravtansine CD19 Conatumumab TRAIL-R2(CD262) Dacetuzumab CD40 Dalotuzumab IGF-1R Dalotuzumab insulin-likegrowth factor I receptor Daratumumab CD38 (cyclic ADP ribose hydrolase)Demcizumab DLL4 Denintuzumab mafodotin CD19 Denosumab RANKLDepatuxizumab EGFR (HER1/ERBB1) Derlotuximab histone complex Detumomabunknown (B-lymphoma cells) Dinutuximab B4GALNT1 Drozitumab TRAIL-R2(CD262) Duligotumab HER3 (ERBB3) Duligotuzumab EGFR (HER1/ERBB1)Dusigitumab ILGF2 Ecromeximab GD3 ganglioside Edrecolomab EpCAMElgemtumab ERBB3 Elotuzumab SLAMF7 (CD319) Elsilimomab IL-6 EmactuzumabCSF1R Emibetuzumab HGFR Emibetuzumab MET Enavatuzumab TNFRSF12AEnfortumab vedotin AGS-22M6 Enoticumab DLL4 Ensituximab MUC5ACEpitumomab cituxetan MUC1 (CD227) Epratuzumab CD22 Ertumaxomab HER2(ERBB2/neu) × CD3 Etaracizumab integrin α5β3 Faralimomab IFNA1Farletuzumab FOLR1 alpha FBTA CD20 × CD3 Ficlatuzumab HGFR FigitumumabIGF-1R Flanvotumab TYRP1(glycoprotein 75) Fresolimumab TGF-β FutuximabEGFR (HER1/ERBB1) Galiximab CD80 Gantiumab IGF-1R Gemtuzumab ozogamicinCD33 Girentuximab Carbonic anhydrase 9 (CA9/CAIX) Glembatumumab vedotinGPNMB glycooptimized trastuzumab-GEX HER2 (ERBB2/neu) Ibritumomabtiuxetan CD20 Icrucumab VEGFR-1 Igovomab MUC16 IMAB362 Claudin-18(CLDN18.2) Imgatuzumab EGFR (HER1/ERBB1) Indatuximab ravtansine SDC1Indusatumab vedotin GUCY2C inebilizumab CD19 Inotuzumab ozogamicin CD22Intetumumab CD51 Iratumumab CD30 (TNFRSF8) Isatuximab CD38 LabetuzumabCEA Lenzilumab CSF2 Lexatumumab TRAIL-R2 (CD262) Lifastuzumab vedotinNaPi2B Lilotomab satetraxetan CD37 Lintuzumab CD33 Lorvotuzumabmertansine CD56 Lucatumumab CD40 Lumiliximab CD23 (IgE receptor)Lumretuzumab ERBB3 Mapatumumab TRAIL-R1 (CD261) Margetuximab HER2(ERBB2/neu) Matuzumab EGFR (HER1/ERBB1) Mepolizumab IL-5 MilatuzumabCD74 Minretumomab TAG-72 Mirvetuximab soravtansine FOLR1 alpha MitumomabGD3 (ganglioside) Mogamulizumab CCR4 Moxetumomab pasudotox CD22Nacolomab tafenatox C242 antigen Naptumomab estafenatox 5T4 NarnatumabRON Necitumumab EGFR (HER1/ERBB1) Nesvacumab ANGPT2 (angiopoietin 2)Nimotuzumab EGFR (HER1/ERBB1) Nofetumomab merpentan EpCAM binutuzumabCD20 Ocaratuzumab CD20 Ofatumumab CD20 Olaratumab PDGFRα Onartuzumab METOntuxizumab CD248 (TEM1) Oportuzumab monatox EpCAM Oregovomab CA-125Otlertuzumab CD37 Panitumumab EGFR (HER1/ERBB1) Pankomab MUC1 (tumorspecific glycosylation) Parsatuzumab EGFL7 Pasotuxizumab FOLH1Patritumab HER3 (ERBB3) Pemtumomab MUC1 (CD227) Pertuzumab HER2(ERBB2/neu) Pinatuzumab vedotin CD22 Pintumomab adenocarcinoma antigenPolatuzumab vedotin CD79B Racotumomab NGcGM3 Radretumab EDB (fibronectinextra domain-B) Ramucirumab VEGFR2 Rilotumumab HGFR Rituximab CD20Robatumumab IGF-1R Sacituzumab govitecan Trop-2 (tumor-associatedcalcium signal transducer 2/EGP-1) Samalizumab CD200 (OX-2 membraneglycoprotein) Satumomab pendetide TAG-72 Seribantumab ERBB3 SeribantumabHER3 (ERBB3) SGN-CDA CD19 SGN-CDA CD33 Sibrotuzumab FAP Siltuximab IL-6Simtuzumab LOXL2 Sofituzumab vedotin CA 125 Solitomab EpCAM SonepcizumabS1P (sphingosine-1-phosphate) Tacatuzumab tetraxetan AFP(alpha-fetoprotein) Taplitumomab paptox CD19 Tarextumab Notch receptorTenatumomab TN-C (tenascin C) Teprotumumab CD221 Tetulomab CD37 TGN CD28Tigatuzumab TRAIL-R2 (CD262) Lebrikizumab IL-13 Tocilizumab IL-6RTositumomab CD20 Tovetumab CD140a Tovetumab PDGFRα Trastuzumab HER2(ERBB2/neu) Trastuzumab emtansine HER2 (ERBB2/neu) TRBS GD2 Tucotuzumabcelmoleukin EpCAM ublituximab CD20 Ublituximab MS4A1 Ulocuplumab CXCR4Vandortuzumab vedotin STEAP1 Vantictumab FZD7 Vanucizumab Ang-2(angiopoietin 2) × VEGF-A Veltuzumab CD20 Vesencumab NRP1 Volociximabintegrin α5β1 Votumumab CTAA16.88 Zalutumumab EGFR (HER1/ERBB1)Zanolimumab CD4 Zatuximab HER1 (EGFR/ERBB1)

Preferably, the neutralizing antibody is chosen from the list ofanti-IL-10 and anti-TGFbeta.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least oneantibody, preferably anti-CD73 antibody or at least one decoy receptoras defined above, or a fragment or variant thereof.

Furthermore, the at least one antibody may preferably chosen fromanti-CD73 antibodies or fragments or variants thereof.

In a further particularly preferred embodiment the at least one antibodyis chosen from an antibody directed against CCR5/CD195 or from anantibody directed against its ligand CCL5/RANTES or fragments orvariants thereof.

In a particularly preferred embodiment the decoy receptor is a solubleCCR5 (chemokine receptor type 5, also known as CD195).

In a particularly preferred embodiment the dominant negative receptor isdominant negative CCR5 (chemokine receptor type 5, also known as CD195).

13. Inhibitors of Myeloid Derived Suppressor Cells (MDSCs)

In this context protein-based inhibitors of myeloid derived suppressorcells (MDSCs), or fragments and variants thereof as disclosed above inthe context of “coding RNA” may be used as additional pharmaceuticallyactive component. Alternatively, nucleic acids encoding these proteinsor peptides or fragments or variants thereof may be used as additionalpharmaceutically active component.

It is particularly preferred to use anti IL-17 antibodies and IL-12 asinhibitors of MDSCs.

In the context of the invention, MDSC inhibition can be achieved bydirect deactivation of MDSCs (e.g., chemical NO inhibitors (PDE-5inhibitors, NO-aspirins, L-NAME), Arginase inhibitors (PDE-5 inhibitors,COX2 inhibitors, NOHA, L-NAME), ROS inhibitors (syntheticTriterpenoids)), by blocking differentiation of MDSCs into mature cells(e.g., ATRA, Vitamin A, Vitamin D3, CpG), by blocking the celldevelopment of MDSCs (e.g. bisphosphorates (zolodronic acid), modulatorsof cell signaling (JAK2/STAT3 inhibitors, Multi-Kinase inhibitors, VEGFinhibitors)), or by depletion of MDSCs (e.g., cytotoxic agents(gemcitabine, cisplatin, paclitaxel, 5-fluorouracil) or HSP 90inhibitors (17-DMAG)). Therefore these compounds may also be used asadditional pharmaceutically active compound.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least oneinhibitor of MDSCs as defined above, or a fragment or variant thereof.

14. IDO Pathway Inhibitors

In this context protein-based IDO pathway inhibitors, or fragments andvariants thereof as disclosed above in the context of “coding RNA” maybe used as additional pharmaceutically active component. Alternatively,nucleic acids encoding these proteins or peptides or fragments orvariants thereof may be used as additional pharmaceutically activecomponent.

Chemical IDO Pathway Inhibitor:

In a further preferred embodiment the additional pharmaceutically activecomponent may be selected from an IDO pathway inhibitor, which ispreferably selected from small molecule inhibitor. Preferably the IDOpathway inhibitor is chosen from the following list: Indoximod (the Disomer of 1-methyl-tryptophan) and NLG919).

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least one IDOpathway inhibitor as defined above, or a fragment or variant thereof.

15. Proteins or Peptides that Bind Inhibitors of Apoptosis

Apoptosis is a tightly regulated cellular process and faulty regulationof apoptosis is a hallmark of human cancers. Targeting key apoptosisregulators with the goal to restore apoptosis in tumor cells has beenpursued as a new cancer therapeutic strategy. XIAP, cIAP1, and cIAP2,members of inhibitor of apoptosis (IAP) proteins, are criticalregulators of cell death and survival and are attractive targets for newcancer therapy. The SMAC/DIABLO protein is an endogenous antagonist ofXIAP, cIAP1, and cIAP2. In the last decade, intense research effortshave resulted in the design and development of several small-moleculeSMAC mimetics now in clinical trials for cancer treatment

In a further preferred embodiment, the inventive composition comprisesat least one molecule that binds inhibitors of apoptosis proteins (IAPB)and thus sensitize cancer cells to apoptotic death.

Therefore it is particularly preferred that the inventive RNA containingcomposition comprises at least one molecule that binds inhibitors ofapoptosis, such as SMAC mimetics.

Particularly preferred proteins or peptides that bind IAPB according tothe present invention comprise Omi/HtrA2, Smac, Smac derived peptides,Smac/DIABLO, and XAF1 (XIAP-associated factor 1) and fragments orvariants thereof.

In this context proteins or peptides that bind inhibitors of apoptosis,or fragments and variants thereof as disclosed above in the context of“coding RNA” may be used as additional pharmaceutically activecomponent. Alternatively, nucleic acids encoding these proteins orpeptides or fragments or variants thereof may be used as additionalpharmaceutically active component.

Therefore it is particularly preferred that the additionalpharmaceutically active component is selected from proteins or peptidesthat bind inhibitors of apoptosis, such as SMAC mimetics. Furthermore itis particularly preferred that such SMAC mimetics used as additionalpharmaceutically active component are small molecules inhibitinginhibitors of apoptosis.

In a particularly preferred embodiment, the at least one RNA of the RNAcontaining composition is an immunostimulating RNA, preferably accordingto SEQ ID NOs. 5, 394, or 10072 and is combined with at least oneproteins or peptides that bind inhibitors of apoptosis as defined above,or a fragment or variant thereof.

16. Anti-Bacterial Agent:

According to the present invention, the at least one additionalpharmaceutically active component which may be contained in theinventive composition, and/or which may be co-administered, may be ananti-bacterial agent. In this context, any anti-bacterial agents knownto one of skill in the art may be used in combination with thecomponents of the inventive composition as defined herein. Non-limitingexamples of anti-bacterial agents include Amikacin, Amoxicillin,Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin,Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin,Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin,Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime,Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefbirome,Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam,Cefbrozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole,Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin,Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin,Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin,Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin,Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin,Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid,Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem,Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin,Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem,Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline,Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin,Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin,Penicillin V, Piperacillin, Piperacillin-sulbactam,Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin,Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole,Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin,Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin,Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, andVoriconazole.

17. Anti-Viral Agents:

According to the present invention, the at least one additionalpharmaceutically active component/compound, which may be contained inthe inventive composition, and/or which may be co-administered, may bean anti-viral agents, preferably, but are not limited to, nucleosideanalogs (e.g., zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine,peramivir, rimantadine, saquinavir, indinavir, ritonavir,alpha-interferons and other interferons, AZT, t-705, zanamivir(Relenza®), and oseltamivir)(Tamiflu®). Other anti-viral agents includeinfluenza virus vaccines, e.g., Fluarix® (Glaxo SmithKline), FluMist®(MedImmune Vaccines), Fluvirin® (Chiron Corporation), Flulaval®(GlaxoSmithKline), Afluria® (CSL Biotherapies Inc.), Agriflu® (Novartis)or Fluzone® (Aventis Pasteur).

18. Drugs:

In some embodiments, the additional pharmaceutically activecomponent/compound may include at least one drug. The term “drug” isintended to include any substance that, when introduced or absorbed intothe body of a living organism, alters normal bodily or cellularfunction. Some non-limiting examples of suitable drugs, includingcombinations and alternative forms of the drugs such as alternative saltforms, free acid form, free base forms, pro-drugs and hydrates, include:analgesics/antipyretics (e.g., aspirin, acetaminophen, ibuprofen,naproxen sodium, buprenorphine, propoxyphene hydrochloride, propoxyphenenapsylate, meperidine hydrochloride, hydromorphone hydrochloride,morphine, oxycodone, codeine, dihydrocodeine bitartrate, pentazocine,hydrocodone bitartrate, levorphanol, diflunisal, trolamine salicylate,nalbuphine hydrochloride, mefenamic acid, butorphanol, cholinesalicylate, butalbital, phenyltoloxamine citrate, diphenhydraminecitrate, methotrimeprazine, cinnamedrine hydrochloride, andmeprobamate); antiasthmatics (e.g., ketotifen and traxanox); antibiotics(e.g., neomycin, streptomycin, chloramphenicol, cephalosporin,ampicillin, penicillin, tetracycline, and ciprofloxacin);antidepressants (e.g., nefopam, oxypertine, doxepin, amoxapine,trazodone, amitriptyline, maprotiline, phenelzine, desipramine,nortriptyline, tranylcypromine, fluoxetine, imipramine, imipraminepamoate, isocarboxazid, trimipramine, and protriptyline); antidiabetics(e.g., biguanides and sulfonylurea derivatives); antifungal agents(e.g., griseofulvin, ketoconazole, itraconazole, amphotericin B,nystatin, and candicidin); antihypertensive agents (e.g., propanolol,propafenone, oxyprenolol, nifedipine, reserpine, trimethaphan,phenoxybenzamine, pargyline hydrochloride, deserpidine, diazoxide,guanethidine monosulfate, minoxidil, rescinnamine, sodium nitroprusside,rauwolfia serpentine, alseroxylon, and phentolamine);anti-inflammatories (e.g., (non-steroidal) indomethacin, ketoprofen,flurbiprofen, naproxen, ibuprofen, ramifenazone, piroxicam, (steroidal)cortisone, dexamethasone, fluazacort, deflazacort, celecoxib, rofecoxib,hydrocortisone, prednisolone, and prednisone); antineoplastics (e.g.,cyclophosphamide, actinomycin, bleomycin, dactinomycin, daunorubicin,doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil,gemcitabine, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin,etoposide, camptothecin and derivatives thereof, phenesterine,paclitaxel and derivatives thereof, docetaxel and derivatives thereof,vinblastine, vincristine, goserelin, leuprolide, tamoxifen, interferonalfa, retinoic acid (ATRA), nitrogen mustard alkylating agents, andpiposulfan); antianxiety agents (e.g., lorazepam, buspirone, prazepam,chlordiazepoxide, oxazepam, clorazepate dipotassium, diazepam,hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol,halazepam, chlormezanone, and dantrolene); immunosuppressive agents(e.g., cyclosporine, azathioprine, mizoribine, and FK506 (tacrolimus));antimigraine agents (e.g., ergotamine, propanolol, isometheptene mucate,and dichloralphenazone); sedatives/hypnotics (e.g., barbiturates such aspentobarbital, pentobarbital, and secobarbital; and benzodiazapines suchas flurazepam hydrochloride, triazolam, and midazolam); antianginalagents (e.g., beta-adrenergic blockers; calcium channel blockers such asnifedipine, and diltiazem; and nitrates such as nitroglycerin,isosorbide dinitrate, pentearythritol tetranitrate, and erythrityltetranitrate); antipsychotic agents (e.g., haloperidol, loxapinesuccinate, loxapine hydrochloride, thioridazine, thioridazinehydrochloride, thiothixene, fluphenazine, fluphenazine decanoate,fluphenazine enanthate, trifluoperazine, chlorpromazine, perphenazine,lithium citrate, and prochlorperazine); antimanic agents (e.g., lithiumcarbonate); antiarrhythmics (e.g., bretylium tosylate, esmolol,verapamil, amiodarone, encamide, digoxin, digitoxin, mexiletine,disopyramide phosphate, procainamide, quinidine sulfate, quinidinegluconate, quinidine polygalacturonate, flecamide acetate, tocamide, andlidocaine); antiarthritic agents (e.g., phenylbutazone, sulindac,penicillanine, salsalate, piroxicam, azathioprine, indomethacin,meclofenamate, gold sodium thiomalate, ketoprofen, auranofin,aurothioglucose, and tolmetin sodium); antigout agents (e.g.,colchicine, and allopurinol); anticoagulants (e.g., heparin, heparinsodium, and warfarin sodium); thrombolytic agents (e.g., urokinase,streptokinase, and alteplase); antifibrinolytic agents (e.g.,aminocaproic acid); hemorheologic agents (e.g., pentoxifylline);antiplatelet agents (e.g., aspirin); anticonvulsants (e.g., valproicacid, divalproex sodium, phenyloin, phenyloin sodium, clonazepam,primidone, phenobarbital, carbamazepine, amobarbital sodium,methsuximide, metharbital, mephobarbital, mephenyloin, phensuximide,paramethadione, ethotoin, phenacemide, secobarbital sodium, clorazepatedipotassium, and trimethadione); antiparkinson agents (e.g.,ethosuximide); antihistamines/antipruritics (e.g., hydroxyzine,diphenhydramine, chlorpheniramine, brompheniramine maleate,cyproheptadine hydrochloride, terfenadine, clemastine fumarate,triprolidine, carbinoxamine, diphenylpyraline, phenindamine, azatadine,tripelennamine, dexchlorpheniramine maleate, methdilazine, and); agentsuseful for calcium regulation (e.g., calcitonin, and parathyroidhormone); antibacterial agents (e.g., amikacin sulfate, aztreonam,chloramphenicol, chloramphenicol palmitate, ciprofloxacin, clindamycin,clindamycin palmitate, clindamycin phosphate, metronidazole,metronidazole hydrochloride, gentamicin sulfate, lincomycinhydrochloride, tobramycin sulfate, vancomycin hydrochloride, polymyxin Bsulfate, colistimethate sodium, and colistin sulfate); antiviral agents(e.g., interferon alpha, beta or gamma, zidovudine, amantadinehydrochloride, ribavirin, and acyclovir); antimicrobials (e.g.,cephalosporins such as cefazolin sodium, cephradine, cefaclor,cephapirin sodium, ceftizoxime sodium, cefoperazone sodium, cefotetandisodium, cefuroxime axetil, cefotaxime sodium, cefadroxil monohydrate,cephalexin, cephalothin sodium, cephalexin hydrochloride monohydrate,cefamandole nafate, cefoxitin sodium, cefonicid sodium, ceforanide,ceftriaxone sodium, ceftazidime, cefadroxil, cephradine, and cefuroximesodium; penicillins such as ampicillin, amoxicillin, penicillin Gbenzathine, cyclacillin, ampicillin sodium, penicillin G potassium,penicillin V potassium, piperacillin sodium, oxacillin sodium,bacampicillin hydrochloride, cloxacillin sodium, ticarcillin disodium,azlocillin sodium, carbenicillin indanyl sodium, penicillin G procaine,methicillin sodium, and nafcillin sodium; macrolides such as,azithromycin, clarithromycin, and erythromycins such as erythromycinethylsuccinate, erythromycin, erythromycin estolate, erythromycinlactobionate, erythromycin stearate, and erythromycin ethylsuccinate;and tetracyclines such as tetracycline hydrochloride, doxycyclinehyclate, and minocycline hydrochloride); anti-infectives (e.g., GM-CSF);bronchodilators (e.g., sympathomimetics such as epinephrinehydrochloride, metaproterenol sulfate, terbutaline sulfate, isoetharine,isoetharine mesylate, isoetharine hydrochloride, albuterol sulfate,albuterol, bitolterolmesylate, isoproterenol hydrochloride, terbutalinesulfate, epinephrine bitartrate, metaproterenol sulfate, epinephrine,and epinephrine bitartrate; anticholinergic agents such as ipratropiumbromide; xanthines such as aminophylline, dyphylline, metaproterenolsulfate, and theophylline; mast cell stabilizers such as cromolynsodium; inhalant corticosteroids such as beclomethasone dipropionate(BDP), and beclomethasone dipropionate monohydrate; salbutamol;ipratropium bromide; budesonide; salmeterol; xinafoate; triamcinolone;nedocromil sodium; flunisolide; fluticasone propionate; steroidalcompounds and hormones (e.g., androgens such as danazol, testosteronecypionate, fluoxymesterone, ethyltestosterone, testosterone enathate,methyltestosterone; estrogens such as estradiol, estropipate, andconjugated estrogens; progestins such as methoxyprogesterone acetate,and norethindrone acetate; corticosteroids such as triamcinolone,betamethasone, betamethasone sodium phosphate, dexamethasone,dexamethasone sodium phosphate, dexamethasone acetate, prednisone,methylprednisolone acetate suspension, triamcinolone acetonide,methylprednisolone, prednisolone sodium phosphate, methylprednisolonesodium succinate, hydrocortisone sodium succinate, triamcinolonehexacetonide, hydrocortisone, hydrocortisone cypionate, prednisolone,fludrocortisone acetate, paramethasone acetate, prednisolone tebutate,prednisolone acetate, prednisolone sodium phosphate, and thyroidhormones such as levothyroxine sodium); hypoglycemic agents (e.g., humaninsulin, purified beef insulin, purified pork insulin, glyburide,metformin, chlorpropamide, glipizide, tolbutamide, and tolazamide);hypolipidemic agents (e.g., clofibrate, dextrothyroxine sodium,probucol, pravastitin, atorvastatin, lovastatin, and niacin); proteins(e.g., DNase, alginase, superoxide dismutase, and lipase); nucleic acids(e.g., anti-sense nucleic acids); agents useful for erythropoiesisstimulation (e.g., erythropoietin); antiulcer/antireflux agents (e.g.,famotidine, cimetidine, and ranitidine hydrochloride);antinauseants/antiemetics (e.g., meclizine hydrochloride, nabilone,prochlorperazine, dimenhydrinate, promethazine hydrochloride,thiethylperazine, and scopolamine); as well as other drugs useful in thecompositions and methods described herein include mitotane,halonitrosoureas, anthrocyclines, ellipticine, ceftriaxone,ketoconazole, ceftazidime, oxaprozin, valacyclovir, urofollitropin,famciclovir, flutamide, enalapril, itraconazole, buspirone, gabapentin,fosinopril, tramadol, acarbose, lorazepam, follitropin, omeprazole,fluoxetine, lisinopril, tramadol, levofloxacin, zafirlukast, interferon,growth hormone, interleukin, erythropoietin, granulocyte stimulatingfactor, nizatidine, bupropion, perindopril, erbumine, adenosine,alendronate, alprostadil, benazepril, betaxolol, bleomycin sulfate,dexfenfluramine, diltiazem, fentanyl, flecamide, gemcitabine, glatirameracetate, granisetron, lamivudine, mangafodipir trisodium, mesalamine,metoprolol fumarate, metronidazole, miglitol, moexipril, monteleukast,octreotide acetate, olopatadine, paricalcitol, somatropin, sumatriptansuccinate, tacrine, verapamil, nabumetone, trovafloxacin, dolasetron,zidovudine, finasteride, tobramycin, isradipine, tolcapone, enoxaparin,fluconazole, lansoprazole, terbinafine, pamidronate, didanosine,diclofenac, cisapride, venlafaxine, troglitazone, fluvastatin, losartan,imiglucerase, donepezil, olanzapine, valsartan, fexofenadine,calcitonin, and ipratropium bromide. In some embodiments, the drug maybe water soluble. In some embodiments, the drug may not be water soluble

19. Combination with Standard Therapy

According to the present invention, the at least one additionalpharmaceutically active component/compound which may be contained in theinventive composition, and/or which may be co-administered, may beselected from any standard therapy used for the treatment of theparticular tumor or cancer disease, e.g any chemotherapy, checkpointmodulator, kinase inhibitor etc.

Adjuvants and Further Components:

According to the present invention, the at least one additionalpharmaceutically active component/compound which may be contained in theinventive composition, and/or which may be co-administered may be anadjuvant. According to a specific embodiment, the inventive compositionmay comprise an adjuvant. In this context, an adjuvant may be understoodas any compound, which is suitable to initiate or increase an immuneresponse of the innate immune system, i.e. a non-specific immuneresponse. With other words, when administered, the inventive compositionpreferably elicits an innate immune response due to the adjuvant,optionally contained therein. Preferably, such an adjuvant may beselected from an adjuvant known to a skilled person and suitable for thepresent case, i.e. supporting the induction of an innate immune responsein a mammal.

Particularly preferred as adjuvants suitable for depot and delivery arecationic or polycationic compounds as defined above for the RNA of theinventive composition as vehicle, transfection or complexation agent.

Furthermore, the inventive composition may comprise one or moreadditional adjuvants which are suitable to initiate or increase animmune response of the innate immune system, i.e. a non-specific immuneresponse, particularly by binding to pathogen-associated molecularpatterns (PAMPs). With other words, when administered, thepharmaceutical composition preferably elicits an innate immune responsedue to the adjuvant, optionally contained therein. Preferably, such anadjuvant may be selected from an adjuvant known to a skilled person andsuitable for the present case, i.e. supporting the induction of aninnate immune response in a mammal.

Also such an adjuvant may be selected from any adjuvant known to askilled person and suitable for the present case, i.e. supporting theinduction of an innate immune response in a mammal and/or suitable fordepot and delivery of the components of the inventive composition.Preferred as adjuvants suitable for depot and delivery are cationic orpolycationic compounds as defined above. Likewise, the adjuvant may beselected from the group consisting of, e.g., cationic or polycationiccompounds as defined above, from chitosan, TDM, MDP, muramyl dipeptide,pluronics, alum solution, aluminium hydroxide, ADJUMERTM(polyphosphazene); aluminium phosphate gel; glucans from algae;algammulin; aluminium hydroxide gel (alum); highly protein-adsorbingaluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF orSPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%),phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAYR1005TM((N-(2-deoxy-2-L-leucylaminob-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOLTM (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAPTM (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of:i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTherTM(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferongamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMSTM; ISCOPREP 7.0.3.TM; liposomes; LOXORIBINETM (7-allyl-8-oxoguanosine); LT 5 oral adjuvant(E. coli labile enterotoxin-protoxin); microspheres and microparticlesof any composition; MF59TM; (squalenewater emulsion); MONTANIDE ISA 51TM(purified incomplete Freund's adjuvant); MONTANIDE ISA 720TM(metabolisable oil adjuvant); MPLTM (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDETM (Nac-Mur-L-Ala-D-Gln-OCH3);MURAPALMITINETM and DMURAPALMITINETM(Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl); NAGO(neuraminidase-galactose oxidase); nanospheres or nanoparticles of anycomposition; NISVs (non-ionic surfactant vesicles); PLEURANTM(β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; microspheres/nanospheres); PLURONIC L121TM; PMMA(polymethylmethacrylate); PODDSTM (proteinoid microspheres);polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylicacid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULONTM(QS-21); Quil-A (Quil-A saponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol);SAF-1TM (“Syntex adjuvant formulation”); Sendai proteoliposomes andSendai containing lipid matrices; Span-85 (sorbitan trioleate); Specol(emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane®(2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Aladipalmitoxypropylamide);Theronyl-MDP (TermurtideTM or [thr 1]-MDP;N-acetylmuramyl-Lthreonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin, microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, 35 IC31, Imidazoquinolines, Ampligen, Ribi529,IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys,Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides,UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable asantagonists including CGRP neuropeptide.

Particularly preferred, an adjuvant may be selected from adjuvants,which support induction of a Th1-immune response or maturation of naïveT-cells, such as GM-CSF, IL-12, IFNg, any RNA as defined herein,preferably an immunostimulatory RNA, CpG DNA, etc.

It is possible that the inventive composition contains besides the atleast one RNA as described above further components which are selectedfrom the group comprising: further antigens or further antigen-providingnucleic acids; a further immunotherapeutic agent; one or more auxiliarysubstances; or any further compound, which is known to beimmunostimulating due to its binding affinity (as ligands) to humanToll-like receptors; and/or an adjuvant nucleic acid, preferably animmunostimulatory RNA (is RNA).

The inventive composition can additionally contain one or more auxiliarysubstances in order to increase its immunogenicity or immunostimulatorycapacity, if desired. A synergistic action of the at least one RNA asdefined herein and of an auxiliary substance, which may be optionallycontained in the inventive composition, is preferably achieved thereby.Depending on the various types of auxiliary substances, variousmechanisms can come into consideration in this respect. For example,compounds that permit the maturation of dendritic cells (DCs), forexample lipopolysaccharides, TNF-alpha or CD40 ligand, form a firstclass of suitable auxiliary substances. In general, it is possible touse as auxiliary substance any agent that influences the immune systemin the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, suchas GM-CFS, which allow an immune response to be enhanced and/orinfluenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that further promote the innate immune response, such asIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32,IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta orTNF-alpha, growth factors, such as hGH.

The inventive composition may contain any further compound, which isknown to be immunostimulating due to its binding affinity (as ligands)to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murineToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13.

Further additives which may be included in the inventive composition areemulsifiers, such as, for example, Tween; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

Pharmaceutical Composition:

In a further aspect, the present invention also provides apharmaceutical composition, comprising the RNA containing composition asdefined herein and a pharmaceutically acceptable carrier and/or vehicle.Preferably the pharmaceutical composition is prepared for intratumoralapplication, preferably by injection into tumor tissue. Sterileinjectable forms of the inventive pharmaceutical composition may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents.

A pharmaceutically acceptable carrier typically includes the liquid ornon-liquid basis of a composition comprising the components of theinventive composition. If the composition is provided in liquid form,the carrier will typically be pyrogen-free water; isotonic saline orbuffered (aqueous) solutions, e.g. phosphate, citrate etc. bufferedsolutions. The injection buffer may be hypertonic, isotonic or hypotonicwith reference to the specific reference medium, i.e. the buffer mayhave a higher, identical or lower salt content with reference to thespecific reference medium, wherein preferably such concentrations of theafore mentioned salts may be used, which do not lead to damage of cellsdue to osmosis or other concentration effects. Reference media are e.g.liquids occurring in “in vivo” methods, such as blood, lymph, cytosolicliquids, or other body liquids, or e.g. liquids, which may be used asreference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds, which are suitable for administration to apatient to be treated, may be used as well for the pharmaceuticalcomposition according to the invention. The term “compatible” as usedhere means that these constituents of the inventive pharmaceuticalcomposition are capable of being mixed with the components of theinventive pharmaceutical composition in such a manner that nointeraction occurs which would substantially reduce the pharmaceuticaleffectiveness of the pharmaceutical composition under typical useconditions.

Administration:

The inventive composition or the inventive pharmaceutical compositionmay be administered by conventional needle injection or needle-free jetinjection into the tumor tissue. In a preferred embodiment the inventivecomposition or the inventive pharmaceutical composition is administeredby jet injection. Jet injection refers to a needle-free injectionmethod, wherein a fluid comprising the inventive composition and,optionally, further suitable excipients is forced through an orifice,thus generating an ultra-fine liquid stream of high pressure that iscapable of penetrating mammalian skin. In principle, the liquid streamforms a hole in the skin, through which the liquid stream is pushed intothe target tissue, namely the tumor tissue. According to the invention,jet injection may be used for intratumoral application of the inventivecomposition.

The inventive composition may be administered by conventional needleinjection or needle-free jet injection adjacent to and/or in closeproximity to the tumor tissue. In a preferred embodiment the inventivepharmaceutical composition is administered by jet injection adjacent toand/or in close proximity to the tumor tissue. Jet injection refers to aneedle-free injection method, wherein a fluid comprising the inventivecomposition and, optionally, further suitable excipients is forcedthrough an orifice, thus generating an ultra-fine liquid stream of highpressure that is capable of penetrating mammalian skin. In principle,the liquid stream forms a hole in the skin, through which the liquidstream is pushed into the target tissue, namely the tumor tissue.According to the invention, jet injection may be used for intratumoralapplication (adjacent to and/or in close proximity to the tumor tissue),particularly for injection of the inventive composition.

In other embodiments, the inventive composition or the inventivepharmaceutical composition may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, intraarticular, intranodal,intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional,intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal,intracardial, intraarterial, and sublingual injection or infusiontechniques.

Further particularly preferred administration routes are intradermal andintramuscular injection.

Despite, the inventive pharmaceutical composition may comprise furthercomponents for facilitating administration and uptake of components ofthe pharmaceutical composition. Such further components may be anappropriate carrier or vehicle, additional adjuvants for supporting anyimmune response, antibacterial and/or antiviral agents.

A further component of the inventive pharmaceutical composition may bean immunotherapeutic agent that can be selected from immunoglobulins,preferably IgGs, monoclonal or polyclonal antibodies, polyclonal serumor sera, etc. Preferably, such a further immunotherapeutic agent may beprovided as a peptide/protein or may be encoded by a nucleic acid,preferably by a DNA or an RNA, more preferably an mRNA.

The inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the RNA molecule(s) as defined herein. Asused herein, a “safe and effective amount” means an amount of the RNAmolecule(s) as defined herein as such that is sufficient tosignificantly induce a positive modification of the tumor or cancerdisease. At the same time, however, a “safe and effective amount” issmall enough to avoid serious side-effects and to permit a sensiblerelationship between advantage and risk. The determination of theselimits typically lies within the scope of sensible medical judgment.

The inventive pharmaceutical composition may be used for human and alsofor veterinary medical purposes, preferably for human medical purposes,as a pharmaceutical composition in general.

Vaccine:

According to another particularly preferred aspect, the inventivecomposition or the inventive pharmaceutical composition may be providedor used as a vaccine. Typically, such a vaccine is as defined above forpharmaceutical compositions. Additionally, such a vaccine typicallycontains the at least one RNA as defined herein or the inventivecomposition comprising a plurality of RNAs. Preferably, the at least oneRNA encodes at least one tumor antigen or at least one immune activatoras defined above. The inventive vaccine may also comprise apharmaceutically acceptable carrier, adjuvant, and/or vehicle as definedherein for the inventive pharmaceutical composition. In the specificcontext of the inventive vaccine, the choice of a pharmaceuticallyacceptable carrier is determined in principle by the manner in which theinventive vaccine is administered. The inventive vaccine may beadministered locally into tumor tissue.

The inventive vaccine can additionally contain one or more auxiliarysubstances in order to increase its immunogenicity or immunostimulatorycapacity, if desired. Particularly preferred are adjuvants as auxiliarysubstances or additives as defined for the pharmaceutical composition.

Kit or Kit of Parts:

In a further aspect, the invention relates to a kit or kit of partscomprising the RNA containing composition as described above, orcomprising the pharmaceutical composition as described above, or thecomponents thereof and optionally technical instructions withinformation on the administration and dosage of the components.

Beside the components of the inventive RNA containing composition, thekit may additionally contain a pharmaceutically acceptable vehicle, anadjuvant and at least one further component e.g. an additionalpharmaceutically active component/compound as defined herein, as well asmeans for administration and technical instructions. The components ofthe composition and possibly further components may be provided inlyophilized form. In a preferred embodiment, prior to use of the kit,the provided vehicle is than added to the lyophilized components in apredetermined amount as written e.g. in the provided technicalinstructions.

Medical Indication:

The present invention furthermore provides several applications and usesof the inventive RNA containing composition, or the pharmaceuticalcomposition, or the vaccine, or the kit or kit of parts as definedherein. As a main aspect of the invention the composition or thepharmaceutical composition or the kit or kit of parts may be used as amedicament, namely for treatment of tumor or cancer diseases. In thiscontext the treatment is preferably done by intratumoral application,especially by injection into tumor tissue. According to another aspect,the present invention is directed to the second medical use of the RNAcontaining composition or the pharmaceutical composition, or thevaccine, or the kit or kit of parts as described above, wherein thesesubject matters are used for preparation of a medicament particularlyfor intratumoral application (administration) for treatment of tumor orcancer diseases.

Preferably, diseases as mentioned herein are selected from tumor orcancer diseases which preferably include e.g. Acute lymphoblasticleukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-relatedcancers, AIDS-related lymphoma, Anal cancer, Appendix cancer,Astrocytoma, Basal cell carcinoma, Bile duct cancer, Bladder cancer,Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstemglioma, Brain tumor, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids,Burkitt lymphoma, childhood Carcinoid tumor, gastrointestinal Carcinoidtumor, Carcinoma of unknown primary, primary Central nervous systemlymphoma, childhood Cerebellar astrocytoma, childhood Cerebralastrocytoma/Malignant glioma, Cervical cancer, Childhood cancers,Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronicmyeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma,Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma,Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors,Childhood Extracranial germ cell tumor, Extragonadal Germ cell tumor,Extrahepatic bile duct cancer, Intraocular melanoma, Retinoblastoma,Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal CarcinoidTumor, Gastrointestinal stromal tumor (GIST), extracranial,extragonadal, or ovarian Germ cell tumor, Gestational trophoblastictumor, Glioma of the brain stem, Childhood Cerebral Astrocytoma,Childhood Visual Pathway and Hypothalamic Glioma, Gastric carcinoid,Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular(liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, childhoodHypothalamic and visual pathway glioma, Intraocular Melanoma, Islet CellCarcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renalcell cancer), Laryngeal Cancer, Leukemias, acute lymphoblastic Leukemia,acute myeloid Leukemia, chronic lymphocytic Leukemia, chronicmyelogenous Leukemia, hairy cell Leukemia, Lip and Oral Cavity Cancer,Liposarcoma, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell LungCancer, Lymphomas, AIDS-related Lymphoma, Burkitt Lymphoma, cutaneousT-Cell Lymphoma, Hodgkin Lymphoma, Non-Hodgkin Lymphomas, PrimaryCentral Nervous System Lymphoma, Waldenström Macroglobulinemia,Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, ChildhoodMedulloblastoma, Melanoma, Intraocular (Eye) Melanoma, Merkel CellCarcinoma, Adult Malignant Mesothelioma, Childhood Mesothelioma,Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer,Childhood Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Diseases, Chronic MyelogenousLeukemia, Adult Acute Myeloid Leukemia, Childhood Acute MyeloidLeukemia, Multiple Myeloma (Cancer of the Bone-Marrow), ChronicMyeloproliferative Disorders, Nasal cavity and paranasal sinus cancer,Nasopharyngeal carcinoma, Neuroblastoma, Oral Cancer, Oropharyngealcancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovariancancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor),Ovarian germ cell tumor, Ovarian low malignant potential tumor,Pancreatic cancer, islet cell Pancreatic cancer, Paranasal sinus andnasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngealcancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma,childhood Pineoblastoma and supratentorial primitive neuroectodermaltumors, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma,Pleuropulmonary blastoma, Primary central nervous system lymphoma,Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer),Cancer of the Renal pelvis and ureter, Retinoblastoma, childhoodRhabdomyosarcoma, Salivary gland cancer, Sarcoma of the Ewing family oftumors, Kaposi Sarcoma, soft tissue Sarcoma, uterine Sarcoma, Sézarysyndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Merkel cellSkin carcinoma, Small intestine cancer, Squamous cell carcinoma,metastatic Squamous neck cancer with occult primary, childhoodSupratentorial primitive neuroectodermal tumor, Testicular cancer,Throat cancer, childhood Thymoma, Thymoma and Thymic carcinoma, Thyroidcancer, childhood Thyroid cancer, Transitional cell cancer of the renalpelvis and ureter, gestational Trophoblastic tumor, Urethral cancer,endometrial Uterine cancer, Uterine sarcoma, Vaginal cancer, childhoodVisual pathway and hypothalamic glioma, Vulvar cancer, Waldenströmmacroglobulinemia, and childhood Wilms tumor (kidney cancer).

Especially preferred examples of tumors or cancers that are suitable forintratumoral administration are prostate cancer, lung cancer, breastcancer, brain cancer, head and neck cancer, thyroid cancer, coloncancer, stomach cancer, liver cancer, pancreas cancer, ovary cancer,skin cancer, urinary bladder, uterus and cervix.

According to a specific embodiment, the medicament may be administeredto the patient as a single dose or as several doses. In certainembodiments, the medicament may be administered to a patient as a singledose followed by a second dose later and optionally even a third, fourth(or more) dose subsequent thereto etc.

Preferably, the inventive composition is provided in an amount of atleast 40 μg RNA per dose. More specifically, the amount of the mRNAcomprised in a single dose is typically at least 200 μg, preferably from200 μg to 1.000 μg, more preferably from 300 μg to 850 μg, even morepreferably from 300 μg to 700 μg.

Treatment with Additional (Pharmaceutical) Compounds:

In a particularly preferred embodiment the subject receiving theinventive composition, or the pharmaceutical composition or vaccine maybe a patient with cancer or tumor who receives or received standardtreatments of cancer. Preferably, the patient has achieved partialresponse or stable disease after having received standard treatments.

The standard treatments of cancer include chemotherapy, radiation,chemoradiation and surgery dependent on the particular cancer or tumortype to be treated, wherein these treatments are applied individually orin combination.

In some embodiments the subject receiving the inventive composition,pharmaceutical composition or vaccine may be a patient with cancer ortumor, who received or receives chemotherapy (e.g. first-line orsecond-line chemotherapy), radiotherapy, chemoradiation (combination ofchemotherapy and radiotherapy), kinase inhibitors, antibody therapyand/or checkpoint modulators (e.g. CTLA4 inhibitors, PD1 pathwayinhibitors), or a patient, who has achieved partial response or stabledisease after having received one or more of the treatments specifiedabove.

In other embodiments the subject receiving the inventive composition,pharmaceutical composition or vaccine may be a patient with cancer ortumor, who received or receives an additional pharmaceutically activecomponent/compound as defined above. Preferably, the subject is apatient, who has achieved partial response or stable disease afterhaving received one or more of the treatments specified above.

According to a further aspect the invention refers to a method oftreatment of tumor or cancer diseases, wherein the RNA containingcomposition as described above, or the pharmaceutical composition asdescribed above, or the vaccine as described above, or the kit or kit ofparts as described above is preferably applied intratumorally,especially by injection into tumor tissue. With respect to furtherfeatures of the method for treatment it is referred to the descriptionabove.

Preferred Intratumoral Applications:

In this context it is particularly preferred that the intratumoralapplication of the RNA containing composition or the pharmaceuticalcomposition or the vaccine as defined above is combined with theapplication of different agents/pharmaceutically activecomponents/compounds. Particularly preferred are antibodies (e.g. checkpoint modulators as e.g. anti-CTLA4, anti-OX40, anti-PD1 or anti-PD-L1)or ligands (e.g. CD40L).

In preferred embodiments the following combinations are particularlypreferred:

-   -   RNAdjuvant (i.t.)+anti-CTLA4 as protein (i.p./i.v.)    -   RNAdjuvant (i.t.)+anti-CTLA4 as protein (i.t.)    -   RNAdjuvant (i.t.)+anti-PD1 as protein (i.p./i.v.)    -   RNAdjuvant (i.t.)+anti-PD1 as protein (i.t.)    -   RNAdjuvant (i.t.)+anti-PD-L1 as protein (i.p./i.v.)    -   RNAdjuvant (i.t.)+anti-PD-L1 as protein (i.t.)    -   RNAdjuvant (i.t.)+CD40L (i.t.) as protein or encoded by a        nucleic acid preferably an RNA, more preferably an mRNA    -   RNAdjuvant (i.t.)+mRNA encoding IL-12+mRNA encoding soluble PD-1        receptor+anti-CD73 (i.p./i.v.)    -   RNAdjuvant (i.t.)+mRNA encoding IL-12+mRNA encoding soluble PD-1        receptor+anti-CD137 (i.p./i.v.)    -   RNadjuvant (i.t.)

(i.t.=intratumoral, i.p.=intraperitoneal, i.v.=intravenous)

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other, wheresuitable. Furthermore, the term “comprising” shall not be narrowlyconstrued as being limited to “consisting of” only, if not specificallymentioned. Rather, in the context of the present invention, “consistingof” is an embodiment specifically contemplated by the inventors to fallunder the scope of “comprising”, wherever “comprising” is used herein.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

The examples and figures shown in the following are merely illustrativeand shall describe the present invention in a further way. These figuresand examples shall not be construed to limit the present inventionthereto.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: shows survival proportions of mice bearing E.G7-OVA tumors afterintratumoral treatment with mRNA encoding IL-12 (IL-12 mRNA) or withrecombinant IL-12 protein (rIL-12 protein). The experiment was performedas described in Example 1. Kaplan-Meier survival curves are presented.

FIG. 2: shows that intratumoral treatment of mice with a combination ofIL-12 mRNA (R2763, SEQ ID NO: 1) and the polymeric carrier cargo complex(R2391, RNAdjuvant®, prepared as described in methods) led to asignificantly decreased tumor volume compared to control groups. Theexperiment was performed as described in Example 2. FIG. 2 shows themean tumor volume at day 21 after tumor challenge (the last day when allanimals were alive). Statistical analysis was performed in GraphPadPrism version 5.04 using Mann Whitney test.

FIG. 3: shows survival proportions of mice bearing CT26 tumors afterintratumoral treatment with a combination of IL-12 mRNA and thepolymeric carrier cargo complex (“RNAdjuvant”) as described in Example 2and in legend of FIG. 2. Kaplan-Meier survival curves are presented.Statistical analysis was performed in GraphPad Prism version 5.04 usingLog-rank test.

FIG. 4: shows survival proportions of mice bearing CT26 tumors afterintratumoral treatment with mRNA encoding the influenza nucleoprotein.The experiment was performed as described in Example 3. Kaplan-Meiersurvival curves are presented.

FIGS. 5A-B: Panel (A) shows an analysis of the median tumor growth ofmice bearing CT26 tumors after intratumoral treatment with mRNA encodingIL-12, RNAdjuvant, and mRNA encoding soluble PD-1. Respectivecombinations of these compounds, including control groups, were testedas indicated in the figure. The experiment was performed as described inExample 4.

-   -   Panel (B) shows survival proportions of mice bearing CT26 tumors        after intratumoral treatment with mRNA encoding IL-12,        RNAdjuvant, and mRNA encoding soluble PD-1. Respective        combinations of these compounds, including control groups, were        tested as indicated in the figure. The experiment was performed        as described in Example 4. Kaplan-Meier survival curves are        presented.

FIG. 6: shows survival proportions of mice bearing CT26 tumors afterintratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNAencoding soluble PD-1 and intraperitoneal treatment of an anti-CD73antibody. Respective combinations of these compounds, including controlgroups, were tested as indicated in the figure. The experiment wasperformed as described in Example 5. Kaplan-Meier survival curves arepresented.

FIG. 7: shows survival proportions of mice bearing CT26 tumors afterintratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNAencoding soluble PD-1 and intraperitoneal treatment of an anti-CD173antibody. Respective combinations of these compounds, including controlgroups, were tested as indicated in the figure. The experiment wasperformed as described in Example 6. Kaplan-Meier survival curves arepresented.

FIG. 8: shows survival proportions of mice bearing CT26 tumors afterintratumoral treatment with RNAdjuvant and intraperitoneal treatment ofan anti-PD-1 antibody. Respective combinations of these compounds,including control groups, were tested as indicated in the figure. Theexperiment was performed as described in Example 7. Kaplan-Meiersurvival curves are presented.

FIG. 9: shows survival proportions of mice bearing CT26 tumors afterintratumoral treatment with mRNA encoding IL-12, RNAdjuvant, mRNAencoding CD40L compared to intratumoral treatment with mRNA encodingIL-12 alone. Respective combinations of these compounds, includingcontrol groups, were tested as indicated in the figure. The experimentwas performed as described in Example 8. Kaplan-Meier survival curvesare presented.

FIG. 10: shows the mRNA sequence R3571 encoding murine CD40L (MmCD40L)according to SEQ ID NO. 10.073

EXAMPLES Methods: Preparation of the RNA

1. Preparation of DNA and RNA Constructs

For the present examples DNA sequences encoding the indicated RNAs (seeTable 17) were prepared and used for subsequent RNA in vitrotranscription reactions.

TABLE 17 RNA constructs RNA Description 5′-UTR 3′-UTR SEQ ID NO. R1328Murine IL-12 encoding mRNA — Muag (3′-UTR of SEQ ID NO:(MmIL-12(GC))-sc-Flag) alpha globin)-A64- 1 C30 R491  mRNA encodingPhotinus — Muag (3′-UTR of SEQ ID NO: pyralis luciferase (pPLuc (GC))alpha globin)-A64- 2 (irrelevant mRNA) C30 R2763 Murine IL-12 encodingmRNA 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: (MmIL-12 (GC)) derived fromA64-C30-histone 3 the ribosomal stem-loop protein 32 L R2244 Luciferaseencoding mRNA 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: (PpLuc(GC)) derivedfrom A64-C30-histone 4 the ribosomal stem-loop protein 32 L R2025Non-coding SEQ ID NO: R2391 immunostimulatory RNA 5 (RNAdjuvant) (SEQ IDNO. 118 of WO2009095226) R2650 mRNA coding for the 5′-TOP-UTRalbumin-3′-UTR- SEQ ID NO: R2651 influenza nucleoprotein derived fromA64-C30-histone 6 (H1N1(PR8)-NP(GC)) the ribosomal stem-loop protein 32L R3971 mRNA encoding solPD-1 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO:derived from A64-C30-histone 389 the ribosomal stem-loop protein 32 LR3571 mRNA encoding murine 5′-TOP-UTR albumin-3′-UTR- SEQ ID NO: CD40L(MmCD40L) derived from A64-C30-histone 10.073 the ribosomal stem-loopprotein 32 L

The constructs of MmIL-12(GC), Influenza NP (GC), soIPD-1 and PpLuc(GC))were prepared by introducing a 5′-TOP-UTR derived from the ribosomalprotein 32L, modifying the wild type coding sequence by introducing aGC-optimized sequence for stabilization, followed by a stabilizingsequence derived from the albumin-3′-UTR, a stretch of 64 adenosines(poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and ahistone stem loop. Most DNA sequences were prepared by modifying thewild type encoding DNA sequences by introducing a GC-optimized sequencefor stabilization, using an in silico algorithm that increase the GCcontent of the respective coding sequence compared to the wild typecoding sequence (in Table 12 indicated as “GC”).

For the present example a DNA sequence encoding the non-codingimmunostimulatory RNA (isRNA) R2025 was prepared and used for subsequentRNA in vitro transcription reactions.

2. RNA In Vitro Transcription

The respective DNA plasmids prepared according to section 1 above weretranscribed in vitro using T7 polymerase. The RNA in vitro transcriptionreactions of the IL-12, the NP, PpLuc, CD40L and soluble PD-1 encodingconstructs were performed in the presence of a CAP analog (m⁷GpppG). TheisRNA R2025 was prepared without CAP analog. Subsequently, the RNA waspurified using PureMessenger® (CureVac, Tübingen, Germany;WO2008/077592A1).

3. Preparation of the Polymeric Cargo Complex (“RNAdjuvant”)

The following cationic peptide as cationic component of the polymericcarrier was used (Cys-Arg₁₂-Cys or CR₁₂C) according to SEQ ID NO: 7.

For synthesis of the polymeric carrier cargo complexes an RNA moleculehaving the RNA sequence R2025 as defined in section 1 above was mixedwith the cationic CR12C peptide component as defined above. Thespecified amount of the RNA was mixed with the respective cationiccomponent in mass ratios as indicated below, thereby forming a complex.If polymerizing cationic components were used according to the presentinvention, polymerization of the cationic components took placesimultaneously to complexation of the nucleic acid cargo. Afterwards,the resulting solution was adjusted with water to a final volume of 50μl and incubated for 30 minutes at room temperature. Further details aredescribed in WO2012013326.

The mass ratio of peptide:RNA was 1:3.7. The polymeric carrier cargocomplex is formed by the disulfide-crosslinked cationic peptide CR12C ascarrier and the immunostimulatory R2025 as nucleic acid cargo. Thispolymeric carrier cargo complex R2025/CR12C (designated R2391) was usedas adjuvant in the following examples (referred to as “RNAdjuvant”)

4. Preparation of the Vaccine Formulation Coding for the InfluenzaNucleoprotein (H1N1(PR8)-NP(GC)) (R2651)

The mRNA (R2650) was complexed with protamine by addition of protamineto the mRNA in the ratio (1:2) (w/w) (adjuvant component). Afterincubation for 10 min, the same amount of free mRNA used asantigen-providing mRNA was added. This vaccine formulation is termedherein R2651 (according to WO2010037539). The vaccine was administeredin Ringer's Lactate solution.

5. Preparation of the RNA for Administration

The naked (that is, non-formulated) PpLuc mRNA (R2244, R491)), IL-12mRNA (R2763, R1328), soluble PD-1 mRNA (R3971), CD40L mRNA (R3571) wereadministered in Ringer's Lactate (RiLa) solution. The co-formulation ofnaked mRNAs and the polymeric carrier cargo complex “RNAdjuvant” (R2391)were also administered in Ringer's Lactate (RiLa) after mixing of bothcomponents directly before injection.

Example 1 Intratumoral Application of mRNA Coding for IL-12

5 female C57BL/6 mice per treatment group were inoculated with 10⁶ cellsE.G7-OVA cells 5 days before the first treatment. For each treatmentgroup 5 established (about 100 mm³) subcutaneously implanted EG.7-OVAtumors were treated. Tumors were treated with 16 μg mRNA coding forMmIL-12 (MmIL-12(GC))-sc-Flag) (R1328) or 0.5 μg MmIL-12 protein on d 0,2, 4, 21, 23 and 25 with 50 μg (1 μg/μl). As control mice were treatedwith an irrelevant mRNA (pPLuc) (R491).

Study day 0 is defined as the first day of treatment. Tumor growth wasmonitored frequently (every 2-3 days). Mice with a volume of >3 cm³ werekilled.

Results of Example 1

FIG. 1 shows that the intratumoral treatment with the mRNA-encoded IL-12(IL-12 mRNA) resulted in a significant increase in survival compared tothe control group. Furthermore an increased survival could be observedcompared to the intratumoral application of recombinant IL-12 protein(rIL-12 protein).

Example 2 Intratumoral Treatment with mRNA Encoding IL-12 in Combinationwith an Immunostimulating RNA (RNAdjuvant®)

The following table 18 summarizes the RNA constructs used for theexample 2.

TABLE 18 RNA constructs for example 2 RNA Description FIG. SEQ ID NO.R2763 Murine IL-12 encoding mRNA 1 SEQ ID NO. 1 R2244 Luciferaseencoding mRNA (PpLuc) 2 SEQ ID NO. 2 R2025 Non-coding immunostimulatoryRNA 3 SEQ ID NO. 3

Balb/c mice (n=6 or 7, see table 14) were injected subcutaneously (s.c.)with 1×10⁶ CT26 cells (colon carcinoma cell line) per mouse (in a volumeof 100 μl PBS) on the right flank on day 0 of the experiment. At day 9after tumor challenge, mice were sorted according to the tumor size toobtain groups with a mean tumor volume of approximately 60 mm³.Intratumoral (i.t.) therapy started at day 9 and continued foradditional 4 injections every 3-4 days. Mice were injected with acombination of mRNA-encoded IL-12 (25 μg of R2763)+RNAdjuvant® (25 μg ofR2391) (group A). To control for local inflammation due to RNAapplication or the injection procedure, mice were injected with controlmRNA coding for luciferase (PpLuc, R2244, group B) or buffer (RiLa,group C), respectively. Untreated mice served as additional control(group D).

Tumor growth was monitored by measuring the tumor size in threedimensions using a calliper. Tumor volume was calculated according tothe following formula:

${{volume}\mspace{14mu} \left( {mm}^{3} \right)} = \frac{{length}\mspace{14mu} ({mm}) \times \pi \times {{width}^{2}\left( {mm}^{2} \right)}}{6}$

On day 9, 11, 14, 17 and 21 of the experiment mice were injectedintratumorally (i.t.) with RNA according to the table 19 below. Thevolume for intratumoral injection was 50 μl.

TABLE 19 Animal groups Strain Number Dose per Route, Group sex of miceRNA mouse volume A BALB/c 7  R2763, 25 μg i.t., Female R2391 of each 50μl RNA B BALB/c 7 R2244 50 μg i.t., Female 50 μl C BALB/c 6 RiLa — i.t.,Female 50 μl D BALB/c 6 — — — Female

Results of Example 2

FIG. 2 shows that the intratumoral treatment with the combination ofmRNA-encoded IL-12 (R2763) and RNAdjuvant® (R2391) resulted in astatistically significant decrease in tumor volume at day 21 after tumorchallenge compared to all control groups.

FIG. 3 shows that the intratumoral treatment with the combination ofmRNA-encoded IL-12 (R2763) and RNAdjuvant® (R2391) resulted in astatistically significant increase in survival compared to all threecontrol groups (group A vs. group B*p=0.0104, group A vs. groupC**p=0.0035, group A vs. Group D*p==0.0263).

Example 3 Vaccination of Mice with mRNA Encoding the InfluenzaNucleoprotein (NP) and Subsequent Intratumoral Treatment withNP-Encoding mRNA

The objective of this experiment was to test whether a pre-existingimmune response can be harnessed against an established tumor. To thisend, mice were first vaccinated with RNActive (vaccine formulationcomplexed with protamine) encoding the influenza nucleoprotein (NP)(R2651) which induces a high level of anti-NP CD8+ T cell responses,then challenged with CT26 tumor cells followed by intratumoral treatmentwith naked RNA encoding NP (R2650).

27 Balb/c mice were vaccinated intradermally (i.d.) with 40 μg ofH1N1(PR8)-NP(GC) RNActive (R2651) (2×50 up or Ringer-Lactate buffer(RiLa) as control on day 0, day 7 and day 16 of the experiment. On day14 all mice were challenged subcutaneously (s.c.) with 1×10⁶ CT26 cellsper mouse (in a volume of 100 μl PBS) on the right flank. On day 22,mice were assigned to the different groups as shown in Table 20.

On day 23, seven days after the second boost, intratumoral (i.t.)application of 50 μg naked H1N1(PR8)-NP(GC) mRNA (R2650) started (onlygroup C) and continued for additional four injections (at day 25, day28, day 31 and day 35). The volume for intratumoral injection was 50 μl.A detailed treatment schedule is shown in Table 21.

Tumor growth was monitored by measuring the tumor size in threedimensions using a calliper. Tumor volume was calculated according tothe following formula:

${{volume}\mspace{14mu} \left( {mm}^{3} \right)} = \frac{{length}\mspace{14mu} ({mm}) \times \pi \times {{width}^{2}\left( {mm}^{2} \right)}}{6}$

TABLE 20 Animal groups Strain Number mRNA Group sex of mice i.d. mRNAi.t. A BALB/c 9 RiLa — Female B BALB/c 9 R2651 — Female (40 μg) C BALB/c9 R2651 R2650 Female (40 μg) (50 μg)

TABLE 21 Vaccination schedule Day Treatment 0 i.d. vaccination allgroups 7 i.d. vaccination all groups 14 Tumor challenge of all groups (1× 10⁶ CT26 cells/mouse) 16 i.d. vaccination all groups 23 i.t.vaccination group C 25,28, 31,35 i.t. vaccination group C

Results of Example 3

FIG. 4 shows that pre-existent immunity (induced in this model by the NPvaccination) increased the median survival time (MST) of mice whichreceived intratumoral application of NP-encoded mRNA compared to micewhich were treated with buffer only (MST=28 vs. MST=21, respectively).

Example 4 Intratumoral Treatment with an Immunostimulating RNA(“RNAdiuvant”) and an mRNA Encoding Soluble PD-1 and an mRNA EncodingIL-12

Table 22 summarizes the treatment as used in the present example.RNAdjuvant and the mRNA constructs encoding IL-12 and soluble PD-1 wereadministered intratumorally (i.t.). In CT26 tumor challenged mice,survival rates and median tumor growth were analyzed.

TABLE 22 Groups, treatment and RNA dilution Nr. of i.t treatmentVaccination Group mice (25 μg for each component schedule A 10 IL-12 +RNAdjuvant + soluble 2× week PD-1 B 10 IL-12 2× week C 10 RNAdjuvant 2×week D 10 RiLa 2× week

Tumor Challenge and Administration of the Inventive Composition:

60 Balb/c mice were challenged subcutaneously with 1×10⁶ CT26 cells permouse (volume in 100 μl PBS) on the right flank on day 0 of theexperiment. On day 8 mice were sorted according to tumor size. Accordingto tumor size, the first vaccination took place on day 8 or 9 (tumorsshould have a size of about 40-50 mm³). Mice were vaccinated withdifferent combinations of mRNAs and RNAdjuvant according to the tableabove. Six vaccinations took place. Volume for intratumoral injectionwas 50 μl.

Mice were injected according to the indicated scheme shown in Table 22.Median tumor growth was determined according to example 3. The resultsof the experiment are shown in FIG. 5, wherein FIG. 5A shows the effectof the inventive composition on tumor growth, and FIG. 5B shows theeffect of the inventive composition on survival.

Results:

The results in FIG. 5A show that the inventive composition comprising anmRNA encoding IL-12 and mRNA encoding soluble PD-1 in combination withRNAdjuvant (group “A” according to Table 22) to strongly decreased themedian tumor volume compared to the other treatments (groups B-Daccording to Table 22). In addition, the results in FIG. 5B show thatthe inventive composition comprising an mRNA encoding IL-12 and mRNAencoding soluble PD-1 in combination with RNAdjuvant (group “A”according to Table 22) strongly increased the survival of tumorchallenged mice compared to the other treatments (groups B-D accordingto Table 22).

Example 5 Intratumoral Treatment with mRNA Encoding IL-12 in Combinationwith an Immunostimulating RNA (“RNAdjuvant”) and mRNA Encoding Sol PD-1and Anti-CD73 Antibody

Table 23 summarizes the treatment as used in the present example. Inaddition to RNAdjuvant and mRNA constructs encoding IL-12 and solublePD-1 (administered intratumorally (i.t.)), an anti CD73 antibody(BioXCell) was co-administered intraperitoreally (i.p.). In CT26 tumorchallenged mice, survival rates were analyzed.

TABLE 23 Groups, treatment and RNA dilution Nr. of i.t. treatment i.p.Vaccination Group mice (25 μg for each component) treatment schedule A10 IL-12 + RNAdjuvant + soluble a-CD73 2× week PD-1 B 10 IL-12 +RNAdjuvant + soluble Rat IgG2a 2× week PD-1 C 10 RiLa a-CD73 2× week D10 RiLa Rat IgG2a 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments(see Example 4).

Mice were injected according to the indicated scheme shown in Table 23.

The results of the experiment are shown in FIG. 6.

Results:

FIG. 6 shows that the intratumoral treatment with mRNA-encoded IL-12(R2763), mRNA encoded sol-PD-1 (R3971) and RNAdjuvant® (R2391) incombination with an i.p. administration of anti CD73 antibody (Group “A”according to Table 23) resulted in a statistically significant increasein survival compared to the relevant control group that only received ananti CD73 antibody (Group “C” according to Table 23) and in an increasein survival rates compared to the treatment withIL-12+RNAdjuvant+soluble PD-1 and a control antibody (Rat IgG2a,BioXCell) (Group “B” according to Table 23).

Example 6 Intratumoral Treatment with mRNA Encoding IL-12 in Combinationwith an Immunostimulating RNA (“RNAdjuvant”) and an Anti-CD137 Antibody

Table 24 summarizes the treatment as used in the present example. Inaddition to RNAdjuvant and the mRNA constructs encoding IL-12 andsoluble PD-1 (administered intratumorally (i.t.)), an anti CD137antibody (BioXCell) was co-administered intraperitoreally (i.p.). InCT26 tumor challenged mice, survival rates were analyzed.

TABLE 24 Groups, treatment and RNA dilution Nr. of i.p. VaccinationGroup mice i.t. treatment (25 μg) treatment schedule A 10 IL-12 +RNAdjuvant + soluble a-CD137 2× week PD-1 B 10 IL-12 + RNAdjuvant +soluble Rat IgG2a 2× week PD-1 C 10 RiLa a-CD137 2× week D 10 RiLa RatIgG2a 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments(see Example 4).

Mice were injected according to the indicated scheme shown in Table 24.

The results of the experiment are shown in FIG. 7.

Results:

FIG. 7 shows that the intratumoral treatment with mRNA-encoded IL-12(R2763) sol-PD-1 (R3971) and RNAdjuvant® (R2391) in combination with ani.p. administration of anti CD-137 antibody (Group “A” according toTable 24) resulted in a significant increase in survival compared to therelevant control group that only received the antibody anti CD-137(Group “C” according to Table 24) and in an increase in survival ratescompared to the treatment with IL-12+RNAdjuvant+soluble PD-1 and acontrol antibody (Rat IgG2a, BioXCell) (Group “B” according to Table24).

Example 7 Treatment with an Immunostimulating RNA (“RNAdjuvant”) inCombination with a Checkpoint Inhibitor Anti PD-1 Antibody

Table 25 summarizes the treatment as used in the present example. Inaddition to RNAdjuvant (administered i.t.), a checkpoint inhibitor antiPD-1 (BioXCell) was administered i.p. In CT26 tumor challenged mice,survival rates were analyzed.

TABLE 25 Groups, treatment and RNA dilution /antibody dilution Amount ofVaccination Group Construct Antibody RNA (μg) schedule A RiLa (i.t.) —2× week B RNAdjuvant Control Ab (i.p.)(100 μg) 25 2× week (i.t.) CRNAdjuvant Anti-PD-1 (i.p.) (200 μg) 25 2× week (i.t.) D RiLa (i.t.)Anti-PD-1 (i.p.) (200 μg) — 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments(see Example 4).

Mice were injected according to the indicated scheme shown in Table 25.

The results of the experiment are shown in FIG. 8.

Results:

FIG. 8 shows that the intratumoral (i.t.) treatment with RNAdjuvant®(R2391) in combination with an i.p. administration of anti PD-1 antibody(Group “C” according to Table 25) resulted in an increase in survivalcompared to the relevant control group that only received the checkpointinhibitor anti PD-1 antibody (Group “D” according to Table 25) and in anincrease in survival rates compared to the treatment with RNAdjuvant anda control antibody (anti hamster IgG, BioXCell) (Group “B” according toTable 25).

Example 8 Intratumoral Treatment with an Immunostimulating RNA(“RNAdjuvant”) and an mRNA Encoding CD40 Ligand (CD40L) and an mRNAEncoding IL-12

Table 26 summarizes the treatment as used in the present example.RNAdjuvant and the mRNA constructs encoding IL-12 and murine CD40L wereadministered intratumorally (i.t.). In CT26 tumor challenged mice,survival rates were analyzed.

TABLE 26 Groups, treatment and RNA dilution Nr. of Vaccination Groupmice i.t. treatment (25 μg per RNA) schedule A 8 IL-12 + RNAdjuvant +CD40L 2× week B 8 IL-12 2× week C 8 RiLa 2× week

Tumor Challenge and Administration of the Inventive Composition:

The tumor challenge was performed according to the previous experiments(see Example 4).

Mice were injected according to the indicated scheme shown in Table 26.

The results of the experiment are shown in FIG. 9.

Results:

The results in FIG. 9 show that the inventive composition comprising anmRNA encoding IL-12 and an mRNA encoding CD40L in combination withRNAdjuvant (group “A” according to table 26) strongly increased themedian survival of tumor challenged mice compared to the othertreatments (groups B-C according to table 26).

1. RNA containing composition comprising at least one RNA for use in thetreatment or prophylaxis of tumor and/or cancer diseases.
 2. The RNAcontaining composition of claim 1, wherein the RNA containingcomposition is to be applied intratumorally, especially by injectioninto tumor tissue.
 3. The RNA containing composition of claim 1, whereinthe at least one RNA is selected from the group consisting of coding RNAand non-coding RNA.
 4. The RNA containing composition of claim 3,wherein the coding RNA comprises at least one coding region encoding atleast one peptide or protein and is preferably selected from the groupconsisting of mRNA, viral RNA, retroviral RNA, and replicon RNA.
 5. TheRNA containing composition of claim 4, wherein the coding RNA is mRNA.6. The RNA containing composition of claim 4, wherein the at least onepeptide or protein is selected or derived from the group consisting ofcytokines, chemokines, suicide gene products, immunogenic proteins orpeptides, apoptosis inducers, angiogenesis inhibitors, heat shockproteins, tumor antigens, β-catenin inhibitors, activators of the STINGpathway, checkpoint modulators, innate immune activators, antibodies,dominant negative receptors and decoy receptors, inhibitors of myeloidderived suppressor cells (MDSCs), IDO pathway inhibitors, and proteinsor peptides that bind inhibitors of apoptosis.
 7. The RNA containingcomposition of claim 6, wherein the cytokine is an interleukin,preferably chosen from the following list: IL-1α, IL-1β, IL-1ra, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28A/B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35.
 8. The RNAcontaining composition of claim 6, wherein the interleukin isinterleukin-12 (IL-12).
 9. The RNA containing composition of claim 6,wherein the cytokine is a member of the TNF family, preferably chosenfrom the following list: TNF, especially TNFα, LTα, LTβ, LIGHT, TWEAK,APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL,TRAIL, RANK ligand.
 10. The RNA containing composition of claim 6,wherein the cytokine is chosen from the following list: FLT3 ligand,G-CSF, GM-CSF, IFNα/β/ω, IFNγ, LIF, M-CSF, MIF, OSM, Stem Cell Factor,TGFβ1, TGFβ2, TGFβ3, TSLP ligand.
 11. The RNA containing composition ofclaim 6, wherein the chemokine is chosen from the following list: CXCL1,CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11,CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1, CCL2, CCL3, CCL4, CCL5,CCL6, CCL7, CCL8, CCL9/10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16,CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26,CCL27, CCL28, XCL1, XCL2, CX3CL1.
 12. The RNA containing composition ofclaim 6, wherein the suicide gene product is a suicide enzyme,preferably a nucleotide metabolizing enzyme.
 13. The RNA containingcomposition of claim 12, wherein the nucleotide metabolizing enzyme ischosen from the following list: thymidine kinase, preferably Herpessimplex virus thymidine kinase, cytosine deaminase, preferably bacterialcytosine deaminase or Yeast cytosine deaminase, deoxynucleoside kinase,preferably Drosophila melanogaster deoxynucleoside kinase, deoxycytidinekinase, preferably a mammalian deoxycytidine kinase, purine nucleosidephosphorylase, preferably a bacterial purine nucleoside phosphorylase.14. The RNA containing composition of claim 6, wherein the at least oneRNA encoding at least one suicide gene product is used in combinationwith a prodrug which is a substrate of the suicide gene product.
 15. TheRNA containing composition of claim 6, wherein the at least one RNAcodes for at least one connexin and at least one suicide gene product.16. The RNA containing composition of claim 6, wherein the RNAcomposition comprises at least one RNA encoding at least one suicidegene product and wherein the RNA composition is used in combination witha further RNA coding for at least one connexin and/or with a protein ofthe connexin family or parts or fragments thereof.
 17. The RNAcontaining composition of claim 6, wherein the immunogenic protein orpeptide is a protein or peptide of a pathogen, more preferably of aviral or bacterial pathogen.
 18. The RNA containing composition of claim17, wherein the immunogenic protein or peptide is at least one proteinor peptide of one virus or bacterium of the following list: influenzavirus type A or B or any other orthomyxovirus (influenza type C),picornaviruses, such as rhinovirus or hepatitis A virus, togaviruses,such as alphavirus or rubivirus, e.g. Sindbis, Semliki-Forest orrubeolavirus, rubella virus, coronaviruses, in particular subtypesHCV-229E or HCV-OC43, rhabdoviruses, such as rabies virus,paramyxoviruses, such as mumps virus, reoviruses, such as group A, B orC rotavirus, hepadnaviruses, such as hepatitis B virus, papoviruses,such as human papillomaviruses of any serotype, adenoviruses, inparticular type 1 to 47, herpesviruses, such as Herpes simplex virus 1,2 or 3, cytomegalovirus, preferably CMVpp65, Epstein Barr virus,vacciniaviruses, the bacterium Chlamydophila pneumoniae, Flaviviruses,such as dengue virus type 1 to 4, yellow fever virus, West Nile virus,Japanese encephalitis virus, hepatitis C virus, caliciviruses,filoviruses, such as Ebola virus, bornaviruses, bunyaviruses, such asRift Valley fever virus, arenaviruses, such as lymphocyticchoriomeningitis virus or hemorrhagic fever viruses, retroviruses, suchas HIV, parvoviruses. 19-61. (canceled)
 62. Method of treatment of tumorand/or cancer diseases with the RNA containing composition as definedaccording to claim 1, preferably by intratumoral application, especiallyby injection into tumor tissue.